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Sample records for gene transfer stability

  1. Histidine-rich stabilized polyplexes for cMet-directed tumor-targeted gene transfer

    NASA Astrophysics Data System (ADS)

    Kos, Petra; Lächelt, Ulrich; Herrmann, Annika; Mickler, Frauke Martina; Döblinger, Markus; He, Dongsheng; Krhač Levačić, Ana; Morys, Stephan; Bräuchle, Christoph; Wagner, Ernst

    2015-03-01

    Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor.Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing

  2. Emergence of collective territorial defense in bacterial communities: horizontal gene transfer can stabilize microbiomes.

    PubMed

    Juhász, János; Kertész-Farkas, Attila; Szabó, Dóra; Pongor, Sándor

    2014-01-01

    Multispecies bacterial communities such as the microbiota of the gastrointestinal tract can be remarkably stable and resilient even though they consist of cells and species that compete for resources and also produce a large number of antimicrobial agents. Computational modeling suggests that horizontal transfer of resistance genes may greatly contribute to the formation of stable and diverse communities capable of protecting themselves with a battery of antimicrobial agents while preserving a varied metabolic repertoire of the constituent species. In other words horizontal transfer of resistance genes makes a community compatible in terms of exoproducts and capable to maintain a varied and mature metagenome. The same property may allow microbiota to protect a host organism, or if used as a microbial therapy, to purge pathogens and restore a protective environment. PMID:24755769

  3. Adenoviral gene transfer into the normal and injured spinal cord: enhanced transgene stability by combined administration of temperature-sensitive virus and transient immune blockade.

    PubMed

    Romero, M I; Smith, G M

    1998-12-01

    This study characterized gene transfer into both normal and injured adult rat dorsal spinal cord using first (E1-/E3-) or second (E1-/E2A125/E3-, temperature-sensitive; ts) generation of replication-defective adenoviral (Ad) vectors. A novel immunosuppressive regimen aimed at blocking CD4/CD45 lymphocytic receptors was tested for improving transgene persistence. In addition, the effect of gene transfer on nociception was also evaluated. Seven days after treatment, numerous LacZ-positive cells were observed after transfection with either viral vector. By 21 days after transfection, beta-galactosidase staining was reduced and suggestive of ongoing cytopathology in both Ad-treated groups, despite the fact that the immunogenicity of LacZ/Adts appeared less when compared with that elicited by the LacZ/Ad vector. In contrast, immunosuppressed animals showed a significant (P < or = 0.05) increase in the number of LacZ-positive cells not displaying cytopathology. In these animals, a concomitant reduction in numbers of macrophages/microglia and CD4 and CD8 lymphocytes was observed. Only animals that received LacZ/Adts and immunosuppression showed transgene expression after 60 days. Similar results were observed in animals in which the L4-L5 dorsal roots were lesioned before transfection. Gene transfer into the dorsal spinal cord did not affect nociception, independent of the adenovirus vector. These results indicate that immune blockade of the CD4/CD45 lymphocytic receptors enhanced transgene stability in adult animals with normal or injured spinal cords and that persistent transgene expression in the spinal cord does not interfere with normal neural function. PMID:10023440

  4. 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. PMID:15761667

  5. 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

  6. Gene transfer for erythropoiesis enhancement.

    PubMed

    Naffakh, N; Danos, O

    1996-08-01

    The spectrum of anemias treated with recombinant human erythropoietin is rapidly broadening. Lifelong treatment with very high doses is now under evaluation for beta-thalassemia and sickle cell anemia. These indications make it worthwhile to search for methods that will allow a permanent systemic delivery of the hormone. Here, we review experimental gene-transfer-based procedures for erythropoietin delivery in vivo. In mice, both ex vivo and direct in vivo approaches for gene transfer have resulted in the long-term production of therapeutic levels of the hormone. Gene transfer of erythropoietin could become a viable alternative to the injection of the purified recombinant protein once reliable procedures for controlling transgene expression are available. PMID:8796920

  7. 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

  8. Gene transfer in intact animals

    NASA Astrophysics Data System (ADS)

    Cline, M. J.; Stang, H.; Mercola, K.; Morse, L.; Ruprecht, R.; Browne, J.; Salser, W.

    1980-04-01

    Resistance to methotrexate was induced in bone marrow cells of mice by transformation in vitro with DNA from a drug-resistant cell line. Transformed cells were injected in vivo and haematopoietic cells expressing resistance were selected by drug treatment of recipients. Transformed cells had elevated levels of dihydrofolate reductase and demonstrated a proliferative advantage over untransformed cells, indicating successful gene transfer.

  9. Horizontal gene transfer in plants.

    PubMed

    Gao, Caihua; Ren, Xiaodong; Mason, Annaliese S; Liu, Honglei; Xiao, Meili; Li, Jiana; Fu, Donghui

    2014-03-01

    Horizontal gene transfer (HGT) describes the transmission of genetic material across species boundaries. HGT often occurs in microbic and eukaryotic genomes. However, the pathways by which HGTs occur in multicellular eukaryotes, especially in plants, are not well understood. We systematically summarized more than ten possible pathways for HGT. The intimate contact which frequently occurs in parasitism, symbiosis, pathogen, epiphyte, entophyte, and grafting interactions could promote HGTs between two species. Besides these direct transfer methods, genes can be exchanged with a vector as a bridge: possible vectors include pollen, fungi, bacteria, viruses, viroids, plasmids, transposons, and insects. HGT, especially when involving horizontal transfer of transposable elements, is recognized as a significant force propelling genomic variation and biological innovation, playing an important functional and evolutionary role in both eukaryotic and prokaryotic genomes. We proposed possible mechanisms by which HGTs can occur, which is useful in understanding the genetic information exchange among distant species or distant cellular components. PMID:24132513

  10. 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

  11. 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.

  12. The stability and degradation of dietary DNA in the gastrointestinal tract of mammals: implications for horizontal gene transfer and the biosafety of GMOs.

    PubMed

    Rizzi, Aurora; Raddadi, Noura; Sorlini, Claudia; Nordgrd, Lise; Nielsen, Kaare Magne; Daffonchio, Daniele

    2012-01-01

    The fate of dietary DNA in the gastrointestinal tract (GIT) of animals has gained renewed interest after the commercial introduction of genetically modified organisms (GMO). Among the concerns regarding GM food, are the possible consequences of horizontal gene transfer (HGT) of recombinant dietary DNA to bacteria or animal cells. The exposure of the GIT to dietary DNA is related to the extent of food processing, food composition, and to the level of intake. Animal feeding studies have demonstrated that a minor amount of fragmented dietary DNA may resist the digestive process. Mammals have been shown to take up dietary DNA from the GIT, but stable integration and expression of internalized DNA has not been demonstrated. Despite the ability of several bacterial species to acquire external DNA by natural transformation, in vivo transfer of dietary DNA to bacteria in the intestine has not been detected in the few experimental studies conducted so far. However, major methodological limitations and knowledge gaps of the mechanistic aspects of HGT calls for methodological improvements and further studies to understand the fate of various types of dietary DNA in the GIT. PMID:22059960

  13. 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

  14. Horizontal gene transfer between bacteria and animals.

    PubMed

    Dunning Hotopp, Julie C

    2011-04-01

    Horizontal gene transfer is increasingly described between bacteria and animals. Such transfers that are vertically inherited have the potential to influence the evolution of animals. One classic example is the transfer of DNA from mitochondria and chloroplasts to the nucleus after the acquisition of these organelles by eukaryotes. Even today, many of the described instances of bacteria-to-animal transfer occur as part of intimate relationships such as those of endosymbionts and their invertebrate hosts, particularly insects and nematodes, while numerous transfers are also found in asexual animals. Both of these observations are consistent with modern evolutionary theory, in particular the serial endosymbiotic theory and Muller's ratchet. Although it is tempting to suggest that these particular lifestyles promote horizontal gene transfer, it is difficult to ascertain given the nonrandom sampling of animal genome sequencing projects and the lack of a systematic analysis of animal genomes for such transfers. PMID:21334091

  15. High expression hampers horizontal gene transfer.

    PubMed

    Park, Chungoo; Zhang, Jianzhi

    2012-01-01

    Horizontal gene transfer (HGT), the movement of genetic material from one species to another, is a common phenomenon in prokaryotic evolution. Although the rate of HGT is known to vary among genes, our understanding of the cause of this variation, currently summarized by two rules, is far from complete. The first rule states that informational genes, which are involved in DNA replication, transcription, and translation, have lower transferabilities than operational genes. The second rule asserts that protein interactivity negatively impacts gene transferability. Here, we hypothesize that high expression hampers HGT, because the fitness cost of an HGT to the recipient, arising from the 1) energy expenditure in transcription and translation, 2) cytotoxic protein misfolding, 3) reduction in cellular translational efficiency, 4) detrimental protein misinteraction, and 5) disturbance of the optimal protein concentration or cell physiology, increases with the expression level of the transferred gene. To test this hypothesis, we examined laboratory and natural HGTs to Escherichia coli. We observed lower transferabilities of more highly expressed genes, even after controlling the confounding factors from the two established rules and the genic GC content. Furthermore, expression level predicts gene transferability better than all other factors examined. We also confirmed the significant negative impact of gene expression on the rate of HGTs to 127 of 133 genomes of eubacteria and archaebacteria. Together, these findings establish the gene expression level as a major determinant of horizontal gene transferability. They also suggest that most successful HGTs are initially slightly deleterious, fixed because of their negligibly low costs rather than high benefits to the recipient. PMID:22436996

  16. Horizontal gene transfer, genome innovation and evolution.

    PubMed

    Gogarten, J Peter; Townsend, Jeffrey P

    2005-09-01

    To what extent is the tree of life the best representation of the evolutionary history of microorganisms? Recent work has shown that, among sets of prokaryotic genomes in which most homologous genes show extremely low sequence divergence, gene content can vary enormously, implying that those genes that are variably present or absent are frequently horizontally transferred. Traditionally, successful horizontal gene transfer was assumed to provide a selective advantage to either the host or the gene itself, but could horizontally transferred genes be neutral or nearly neutral? We suggest that for many prokaryotes, the boundaries between species are fuzzy, and therefore the principles of population genetics must be broadened so that they can be applied to higher taxonomic categories. PMID:16138096

  17. 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.

  18. 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

  19. In vitro gene transfer by electrosonoporation.

    PubMed

    Escoffre, J M; Kaddur, K; Rols, M P; Bouakaz, A

    2010-10-01

    Among the nonviral methods for gene delivery in vitro, electroporation is simple, inexpensive and safe. To upregulate the expression level of transfected gene, we investigated the applicability of electrosonoporation. This approach consists of a combination of electric pulses and ultrasound assisted with gas microbubbles. Cells were first electroporated with plasmid DNA encoding-enhanced green fluorescent protein and then sonoporated in presence of contrast microbubbles. Twenty-four hours later, cells that received electrosonoporation demonstrated a four-fold increase in transfection level and a six-fold increase in transfection efficiency compared with cells having undergone electroporation alone. Although electroporation induced the formation of DNA aggregates into the cell membrane, sonoporation induced its direct propulsion into the cytoplasm. Sonoporation can improve the transfer of electro-induced DNA aggregates by allowing its free and rapid entrance into the cells. These results demonstrated that in vitro gene transfer by electrosonoporation could provide a new potent method for gene transfer. PMID:20850028

  20. 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

  1. Stabilizing gene regulatory networks through feedforward loops

    NASA Astrophysics Data System (ADS)

    Kadelka, C.; Murrugarra, D.; Laubenbacher, R.

    2013-06-01

    The global dynamics of gene regulatory networks are known to show robustness to perturbations in the form of intrinsic and extrinsic noise, as well as mutations of individual genes. One molecular mechanism underlying this robustness has been identified as the action of so-called microRNAs that operate via feedforward loops. We present results of a computational study, using the modeling framework of stochastic Boolean networks, which explores the role that such network motifs play in stabilizing global dynamics. The paper introduces a new measure for the stability of stochastic networks. The results show that certain types of feedforward loops do indeed buffer the network against stochastic effects.

  2. Unsupervised learning in detection of gene transfer.

    PubMed

    Hamel, L; Nahar, N; Poptsova, M S; Zhaxybayeva, O; Gogarten, J P

    2008-01-01

    The tree representation as a model for organismal evolution has been in use since before Darwin. However, with the recent unprecedented access to biomolecular data, it has been discovered that, especially in the microbial world, individual genes making up the genome of an organism give rise to different and sometimes conflicting evolutionary tree topologies. This discovery calls into question the notion of a single evolutionary tree for an organism and gives rise to the notion of an evolutionary consensus tree based on the evolutionary patterns of the majority of genes in a genome embedded in a network of gene histories. Here, we discuss an approach to the analysis of genomic data of multiple genomes using bipartition spectral analysis and unsupervised learning. An interesting observation is that genes within genomes that have evolutionary tree topologies, which are in substantial conflict with the evolutionary consensus tree of an organism, point to possible horizontal gene transfer events which often delineate significant evolutionary events. PMID:18509479

  3. [Synthesis of new gene-loaded microbubbles serve as gene delivery vehicle applied in reporter gene transfer into cardiac myocytes].

    PubMed

    Wang, Guozhong; Hu, Shenjiang; Zheng, Zhelan; Sun, Jian; Zheng, Xia; Zhu, Zhaohui; Li, Jiang; Yao, Yumei

    2006-08-01

    To improve the stability and gene-carried capability of gene-attached microbubbles, the method for manufacture of albumin microbubbles was modified and new gene-loaded microbubbles were synthesized by incorporated gene-PEI complex into the shell of microbubbles. Agarose gel electrophoresis and bacteria transformation showed that PEI had the ability to provide the protection of plasmid DNA from ultrasonic degradation. The new gene-loaded microbubbles exhibited excellent acoustical and hemorheological properties. Moreover, they could carry more plasmid DNA than gene-attached microbubbles. beta-galactosidase plasmid transfection into cardiac myocytes was performed by using ultrasound targeted destruction of new gene-loaded microbubbles or gene-attached microbubbles. Gene expression in cardiac myocytes was detected by beta-galactosidase in situ staining and quantitive assay. It was shown that beta-galactosidase activity in cardiac myocytes was enhanced 107-fold by ultrasonic destruction of gene-loaded microbubbles compared with naked plasmid transfection and new gene-loaded microbubbles resulted in 6.85-fold increase in beta-galactosidase activity compared with optimal transfection mediated by gene-attached microbubbles. These results suggested that ultrasonic destruction of the gene-loaded microbubbles can enhance the cardiac myocytes exogenous gene transfer efficiency significantly and new gene-loaded microbubbles is an efficient and safe gene delivery vehicle. PMID:17002125

  4. 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.

  5. 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

  6. 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

  7. Engineering stability in gene networks by autoregulation

    NASA Astrophysics Data System (ADS)

    Becskei, Attila; Serrano, Luis

    2000-06-01

    The genetic and biochemical networks which underlie such things as homeostasis in metabolism and the developmental programs of living cells, must withstand considerable variations and random perturbations of biochemical parameters. These occur as transient changes in, for example, transcription, translation, and RNA and protein degradation. The intensity and duration of these perturbations differ between cells in a population. The unique state of cells, and thus the diversity in a population, is owing to the different environmental stimuli the individual cells experience and the inherent stochastic nature of biochemical processes (for example, refs 5 and 6). It has been proposed, but not demonstrated, that autoregulatory, negative feedback loops in gene circuits provide stability, thereby limiting the range over which the concentrations of network components fluctuate. Here we have designed and constructed simple gene circuits consisting of a regulator and transcriptional repressor modules in Escherichia coli and we show the gain of stability produced by negative feedback.

  8. Engineering stability in gene networks by autoregulation.

    PubMed

    Becskei, A; Serrano, L

    2000-06-01

    The genetic and biochemical networks which underlie such things as homeostasis in metabolism and the developmental programs of living cells, must withstand considerable variations and random perturbations of biochemical parameters. These occur as transient changes in, for example, transcription, translation, and RNA and protein degradation. The intensity and duration of these perturbations differ between cells in a population. The unique state of cells, and thus the diversity in a population, is owing to the different environmental stimuli the individual cells experience and the inherent stochastic nature of biochemical processes (for example, refs 5 and 6). It has been proposed, but not demonstrated, that autoregulatory, negative feedback loops in gene circuits provide stability, thereby limiting the range over which the concentrations of network components fluctuate. Here we have designed and constructed simple gene circuits consisting of a regulator and transcriptional repressor modules in Escherichia coli and we show the gain of stability produced by negative feedback. PMID:10850721

  9. 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

  10. 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

  11. 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

  12. Development of a transfer function method for dynamic stability measurement

    NASA Technical Reports Server (NTRS)

    Johnson, W.

    1977-01-01

    Flutter testing method based on transfer function measurements is developed. The error statistics of several dynamic stability measurement methods are reviewed. It is shown that the transfer function measurement controls the error level by averaging the data and correlating the input and output. The method also gives a direct estimate of the error in the response measurement. An algorithm is developed for obtaining the natural frequency and damping ratio of low damped modes of the system, using integrals of the transfer function in the vicinity of a resonant peak. Guidelines are given for selecting the parameters in the transfer function measurement. Finally, the dynamic stability measurement technique is applied to data from a wind tunnel test of a proprotor and wing model.

  13. 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.

  14. Gene Transfer between Salmonella enterica Serovar Typhimurium inside Epithelial Cells

    PubMed Central

    Ferguson, Gayle C.; Heinemann, Jack A.; Kennedy, Martin A.

    2002-01-01

    Virulence and antibiotic resistance genes transfer between bacteria by bacterial conjugation. Conjugation also mediates gene transfer from bacteria to eukaryotic organisms, including yeast and human cells. Predicting when and where genes transfer by conjugation could enhance our understanding of the risks involved in the release of genetically modified organisms, including those being developed for use as vaccines. We report here that Salmonella enterica serovar Typhimurium conjugated inside cultured human cells. The DNA transfer from donor to recipient bacteria was proportional to the probability that the two types of bacteria occupied the same cell, which was dependent on viable and invasive bacteria and on plasmid tra genes. Based on the high frequencies of gene transfer between bacteria inside human cells, we suggest that such gene transfers occur in situ. The implications of gene transfer between bacteria inside human cells, particularly in the context of antibiotic resistance, are discussed. PMID:11914355

  15. Reducible cationic lipids for gene transfer.

    PubMed Central

    Wetzer, B; Byk, G; Frederic, M; Airiau, M; Blanche, F; Pitard, B; Scherman, D

    2001-01-01

    One of the main challenges of gene therapy remains the increase of gene delivery into eukaryotic cells. We tested whether intracellular DNA release, an essential step for gene transfer, could be facilitated by using reducible cationic DNA-delivery vectors. For this purpose, plasmid DNA was complexed with cationic lipids bearing a disulphide bond. This reduction-sensitive linker is expected to be reduced and cleaved in the reducing milieu of the cytoplasm, thus potentially improving DNA release and consequently transfection. The DNA--disulphide-lipid complexation was monitored by ethidium bromide exclusion, and the size of complexes was determined by dynamic light scattering. It was found that the reduction kinetics of disulphide groups in DNA--lipid complexes depended on the position of the disulphide linker within the lipid molecule. Furthermore, the internal structure of DNA--lipid particles was examined by small-angle X-ray scattering before and after lipid reduction. DNA release from lipid complexes was observed after the reduction of disulphide bonds of several lipids. Cell-transfection experiments suggested that complexes formed with selected reducible lipids resulted in up to 1000-fold higher reporter-gene activity, when compared with their analogues without disulphide bonds. In conclusion, reduction-sensitive groups introduced into cationic lipid backbones potentially allow enhanced DNA release from DNA--lipid complexes after intracellular reduction and represent a tool for improved vectorization. PMID:11389682

  16. Horizontal gene transfer from Agrobacterium to plants

    PubMed Central

    Matveeva, Tatiana V.; Lutova, Ludmila A.

    2014-01-01

    Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named “cellular T-DNA” (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role. PMID:25157257

  17. 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.

  18. 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.

  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

    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).Molecular Therapy - Nucleic Acids (2012) 1, e56; doi:10.1038/mtna.2012.47; published online 27 November 2012. 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. Skyrmion stability in nanocontact spin-transfer oscillators

    SciTech Connect

    Chui, C. P.; Zhou, Yan

    2015-09-15

    We investigate the conditions for nanocontact spin-transfer oscillators (NC-STOs) that allow for stabilization of a skyrmion. Emphasis is made on the breathing mode, which can be regarded as a source of microwave generation. Micromagnetic simulations of NC-STOs with varying parameters have been performed, with the resulting magnetization plotted in the form of phase diagrams. It is found that control of spin wave mode in conventional STOs can be applied to skyrmion-based STOs.

  4. 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.

  5. 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.

  6. 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

  7. Gene transfer system for Rhodopseudomonas viridis.

    PubMed Central

    Lang, F S; Oesterhelt, D

    1989-01-01

    A gene transfer system for Rhodopseudomonas viridis was established which uses conjugation with Escherichia coli S17-I as the donor and mobilizable plasmids as vectors. Initially, plasmids of the incompatibility group P1 (pRK290 and pRK404) were used. The more effective shuttle vectors between E. coli and R. viridis, pKV1 and pKVS1, were derived from plasmid pBR322 and showed the highest conjugation frequency (10(-2] thus far demonstrated in purple bacteria. It was also demonstrated that Rhizobium meliloti can be used as a donor for conjugation with R. viridis. From a genomic cosmid library of R. viridis constructed in the vector pHC79, clones that coded for subunits H (puh operon), L, M and cytochrome c (puf operon) of the photosynthetic reaction center were isolated and characterized. For linkage of the two operons on the genome, cosmids that overlapped with the operon-carrying clones were identified. The relative positions of the two operons could not be determined, but the operons must be more than 100 kilobase pairs apart. Thus, the genomic organization of the reaction center in R. viridis is different from that of Rhodobacter capsulatus, for which a distance of about 39 kilobase pairs was determined. From a spontaneous mutant of R. viridis that is resistant to the herbicide terbutryn, the puf operon was cloned in pKVS1 and transferred by conjugation into R. viridis wild-type cells. The resulting exconjugants were resistant to the herbicide, which demonstrated that the puf operon on pKVS1 constructions was functionally expressed in R. viridis. Images PMID:2666398

  8. Kinetics of conjugative gene transfer on surfaces in granular porous media

    NASA Astrophysics Data System (ADS)

    Massoudieh, A.; Crain, C.; Lambertini, E.; Nelson, K. E.; Barkouki, T.; L'Amoreaux, P.; Loge, F. J.; Ginn, T. R.

    2010-03-01

    The transfer of genetic material among bacteria in the environment can occur both in the planktonic and attached state. Given the propensity of organisms to exist in sessile microbial communities in oligotrophic subsurface conditions, and that such conditions typify the subsurface, this study focuses on exploratory modeling of horizontal gene transfer among surface-associated Escherichiacoli in the subsurface. The mathematics so far used to describe the kinetics of conjugation in biofilms are developed largely from experimental observations of planktonic gene transfer, and are absent of lags or plasmid stability that appear experimentally. We develop a model and experimental system to quantify bacterial filtration and gene transfer in the attached state, on granular porous media. We include attachment kinetics described in Nelson et al. (2007) using the filtration theory approach of Nelson and Ginn (2001, 2005) with motility of E. coli described according to Biondi et al. (1998).

  9. Kinetics of conjugative gene transfer on surfaces in granular porous media

    NASA Astrophysics Data System (ADS)

    Ginn, T.; Massoudieh, A.; Nelson, K.; Mathew, A.; Lambertini, E.

    2005-12-01

    The transfer of genetic material among bacteria in the environment can occur both in the planktonic and attached state. Given the propensity of organisms to exist in sessile microbial communities in oligotrophic conditions, and that such conditions typify the subsurface, this study focuses on exploratory modeling of horizontal gene transfer among surface-associated E. coli in the subsurface. The mathematics so far used to describe the kinetics of conjugation in biofilms are developed largely from experimental observations of planktonic gene transfer, and are absent of lags or plasmid stability that appear experimentally. We develop a model for bacterial filtration and gene transfer in the attached state, for the early stages of biofilm formation using a recently revised filtration theory approach (Nelson and Ginn, 2005) with motility of E. coli described as a continuous time random walk according to data from microflow chamber experiments (Biondi et al., 2002).

  10. Laser frequency stabilization and shifting by using modulation transfer spectroscopy

    NASA Astrophysics Data System (ADS)

    Cheng, Bing; Wang, Zhao-Ying; Wu, Bin; Xu, Ao-Peng; Wang, Qi-Yu; Xu, Yun-Fei; Lin, Qiang

    2014-10-01

    The stabilizing and shifting of laser frequency are very important for the interaction between the laser and atoms. The modulation transfer spectroscopy for the 87Rb atom with D2 line transition F = 2 → F' = 3 is used for stabilizing and shifting the frequency of the external cavity grating feedback diode laser. The resonant phase modulator with electro—optical effect is used to generate frequency sideband to lock the laser frequency. In the locking scheme, circularly polarized pump- and probe-beams are used. By optimizing the temperature of the vapor, the pump- and probe-beam intensity, the laser linewidth of 280 kHz is obtained. Furthermore, the magnetic field generated by a solenoid is added into the system. Therefore the system can achieve the frequency locking at any point in a range of hundreds of megahertz frequency shifting with very low power loss.

  11. 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

  12. Cotransfer of linked eukaryotic genes and efficient transfer of hypoxanthine phosphoribosyltransferase by DNA-mediated gene transfer.

    PubMed Central

    Peterson, J L; McBride, O W

    1980-01-01

    The efficiency of DNA-mediated transfer of the gene (hprt) for hypoxanthine phosphoribosyltransferase (HPRT; IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) is dependent upon the recipient cell used. hprt has been transferred into mouse TG8 or Chinese hamster CHTG49 cells at a high frequency, similar to the frequency of the gene (tk) for thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) transfer into mouse LMTK- cells (i.e., 10(-6)). In contrast, the frequency of transfer of hprt into mouse A9 cells was about two orders of magnitude less. The identification of efficient recipient cells for hprt transfer permits the use of DNA-mediated transfer as a bioassay for the gene. Cotransfer of the linked tk gene and the gene (galk) for galactokinase (ATP: D-galactose 1-phosphotransferase, EC 2.7.1.6) to LMTK- cells has been detected once among 87 tk transferrents. This suggests that the distance between the tk and galk genes in the Chinese hamster genome may be smaller than was previously thought. Significant differences between chromosome-mediated and DNA-mediated gene transfer were observed with respect to both the size of the transferred functional genetic fragment and the recipient cell specificity. Images PMID:6929511

  13. Gene Transfer Strategies to Promote Chondrogenesis and Cartilage Regeneration.

    PubMed

    Im, Gun-Il

    2016-04-01

    Gene transfer has been used experimentally to promote chondrogenesis and cartilage regeneration. While it is controversial to apply gene therapy for nonlethal conditions such as cartilage defect, there is a possibility that the transfer of therapeutic transgenes may dramatically increase the effectiveness of cell therapy and reduce the quantity of cells that are needed to regenerate cartilage. Single or combination of growth factors and transcription factors has been transferred to mesenchymal stem cells or articular chondrocytes using both nonviral and viral approaches. The current challenge for the clinical applications of genetically modified cells is ensuring the safety of gene therapy while guaranteeing effectiveness. Viral gene delivery methods have been mainstays currently with enhanced safety features being recently refined. On the other hand, efficiency has been greatly improved in nonviral delivery. This review summarizes the history and recent update on the gene transfer to enhance chondrogenesis from stem cells or articular chondrocytes. PMID:26414246

  14. 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

  15. 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.

  16. 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

  17. Fibrin-mediated lentivirus gene transfer: implications for lentivirus microarrays

    PubMed Central

    Raut, Shruti; Lei, Pedro; Padmashali, Roshan; Andreadis, Stelios T.

    2010-01-01

    We employed fibrin hydrogel as bioactive matrix for lentivirus mediated gene transfer. Fibrin-mediated gene transfer was highly efficient and exhibited strong dependence on fibrinogen concentration. Efficient gene transfer was achieved with fibrinogen concentration between 3.75 – 7.5 mg/mL. Lower fibrinogen concentrations resulted in diffusion of virus out of the gel while higher concentrations led to ineffective fibrin degradation by target cells. Addition of fibrinolytic inhibitors decreased gene transfer in a dose-dependent manner suggesting that fibrin degradation by target cells may be necessary for successful gene delivery. Under these conditions transduction may be limited only to cells interacting with the matrix thereby providing a method for spatially localized gene delivery. Indeed, when lentivirus-containing fibrin microgels were spotted in an array format gene transfer was confined to virus-containing fibrin spots with minimal cross-contamination between neighboring sites. Collectively, our data suggest that fibrin may provide an effective matrix for spatially-localized gene delivery with potential applications in high-throughput lentiviral microarrays and in regenerative medicine. PMID:20153386

  18. 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...

  19. TWIN BINARIES: STUDIES OF STABILITY, MASS TRANSFER, AND COALESCENCE

    SciTech Connect

    Lombardi, J. C.; Holtzman, W.; Gearity, K.; Dooley, K. L.; Kalogera, V.; Rasio, F. A.

    2011-08-20

    Motivated by suggestions that binaries with almost equal-mass components ('twins') play an important role in the formation of double neutron stars and may be rather abundant among binaries, we study the stability of synchronized close and contact binaries with identical components in circular orbits. In particular, we investigate the dependency of the innermost stable circular orbit on the core mass, and we study the coalescence of the binary that occurs at smaller separations. For twin binaries composed of convective main-sequence stars, subgiants, or giants with low-mass cores (M{sub c} {approx}< 0.15M, where M is the mass of a component), a secular instability is reached during the contact phase, accompanied by a dynamical mass transfer instability at the same or at a slightly smaller orbital separation. Binaries that come inside this instability limit transfer mass gradually from one component to the other and then coalesce quickly as mass is lost through the outer Lagrangian points. For twin giant binaries with moderate to massive cores (M{sub c} {approx}> 0.15M), we find that stable contact configurations exist at all separations down to the Roche limit, when mass shedding through the outer Lagrangian points triggers a coalescence of the envelopes and leaves the cores orbiting in a central tight binary. In addition to the formation of binary neutron stars, we also discuss the implications of our results for the production of planetary nebulae with double degenerate central binaries.

  20. Evolution of and Horizontal Gene Transfer in the Endornavirus Genus

    PubMed Central

    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

  1. 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

  2. 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

  3. Advancements in gene transfer-based therapy for hemophilia A

    PubMed Central

    Doering, Christopher B; Spencer, H Trent

    2010-01-01

    Gene therapy has promised clinical benefit to those suffering with hemophilia A, but this benefit has not yet been realized. However, during the past two decades, basic and applied gene therapy research has progressed and the goal of gene therapy for hemophilia A is once again in our sights. The hemophilia A patient population suffers from a disease that requires invasive, lifelong management, is exorbitantly expensive to treat, has geographically limited treatment access and can become untreatable due to immune reactions to the treatment product. Subsequent to the cloning of the factor VIII gene and cDNA in the early 1980s, academic and commercial research laboratories began to pursue gene transfer-based therapies to supplement or supplant the available protein replacement therapy. However, to date, clinical trials for gene therapy of hemophilia A have been unsuccessful. Three trials have been conducted with each having tested a different gene-transfer strategy and each demonstrating that there is a considerable barrier to achieving sustained expression of therapeutic amounts of factor VIII. Recent progress has been made in gene-transfer technology and, relevant to hemophilia A, towards increasing the biosynthetic efficiency of factor VIII. These advances are now being combined to develop novel strategies to treat and possibly cure hemophilia A. PMID:20577574

  4. 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

  5. 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. PMID:27189546

  6. Experiments on gene transferring to primary hematopoietic cells by liposome.

    PubMed

    Hu, L; Zhang, B

    2000-01-01

    Liposomes have showed many advantages in mediating exogenous gene into many cell types in vitro and in vivo. But few data are available concerning gene transfer into hematopoietic cells. In this report, we described two-marker genes (Neo R and Lac Z) co-transferred into hematopoietic cells of human and mouse by using liposome in vitro. The efficiency of gene transfer was tested by X-gal staining and observation of colony formation. The X-gal blue staining rate of transduced cells was about (13.33 +/- 2.68)% in human and about (16.28 +/- 2.95)% in mouse without G418 selection. After G418 selection, the blue cell rate was (46.06 +/- 3.47)% in human and (43.45 +/- 4.1)% in mouse, which were markedly higher than those before selection, suggesting that high-efficiency gene transfer and expression could be attained in primary hematopoietic cells using this easy and harmless transduction protocol. At the same time, this protocol provided experimental data for clinicians to investigate the biology of marrow reconstitution and trace the origin of relapse after autologous bone marrow transplantation for the patients with leukemia. PMID:12840913

  7. Horizontal gene transfer in eukaryotes: The weak-link model

    PubMed Central

    Huang, Jinling

    2013-01-01

    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. PMID:24037739

  8. Stability and Heat Transfer Characteristics of Condensing Films

    NASA Astrophysics Data System (ADS)

    Hermanson, J. C.; Pedersen, P. C.; Allen, J. S.; Shear, M. A.; Chen, Z. Q.; Alexandrou, A. N.

    2002-11-01

    The overall objective of this research is to investigate the fundamental physics of film condensation in reduced gravity. The condensation of vapor on a cool surface is important in many engineering problems,including spacecraft thermal control and also the behavior of condensate films that may form on the interior surfaces of spacecraft. To examine the effects of body force on condensing films, two different geometries have been tested in the laboratory: (1) a stabilizing gravitational body force (+1g, or condensing surface facing 'upwards') and (2) de-stabilizing gravitational body force (-1g, or 'downwards'). For each geometry, different fluid configurations are employed to help isolate the fluid mechanical and thermal mechanisms operative in condensing films. The fluid configurations are (a) a condensing film, and (b) a non-condensing film with film growth by mass addition by through the plate surface. Condensation experiments are conducted in a test cell containing a cooled copper or brass plate with an exposed diameter of 12.7 cm. The metal surface is polished to allow for double-pass shadowgraph imaging, and the test surface is instrumented with imbedded heat transfer gauges and thermocouples. Representative shadowgraph images of a condensing, unstable (-1g) n-pentane film are shown. The interfacial disturbances associated with the de-stabilizing body force leading to droplet formation and break-off can be clearly seen. The heat transfer coefficient associated with the condensing film is shown. The heat transfer coefficient is seen to initially decrease, consistent with the increased thermal resistance due to layer growth. For sufficiently long time, a steady value of heat transfer is observed, accompanied by continuous droplet formation and break-off. The non-condensing cell consists of a stack of thin stainless steel disks 10 cm in diameter mounted in a brass enclosure. The disks are perforated with a regular pattern of 361 holes each 0.25 mm in diameter

  9. Stability and Heat Transfer Characteristics of Condensing Films

    NASA Technical Reports Server (NTRS)

    Hermanson, J. C.; Pedersen, P. C.; Allen, J. S.; Shear, M. A.; Chen, Z. Q.; Alexandrou, A. N.

    2002-01-01

    The overall objective of this research is to investigate the fundamental physics of film condensation in reduced gravity. The condensation of vapor on a cool surface is important in many engineering problems,including spacecraft thermal control and also the behavior of condensate films that may form on the interior surfaces of spacecraft. To examine the effects of body force on condensing films, two different geometries have been tested in the laboratory: (1) a stabilizing gravitational body force (+1g, or condensing surface facing 'upwards') and (2) de-stabilizing gravitational body force (-1g, or 'downwards'). For each geometry, different fluid configurations are employed to help isolate the fluid mechanical and thermal mechanisms operative in condensing films. The fluid configurations are (a) a condensing film, and (b) a non-condensing film with film growth by mass addition by through the plate surface. Condensation experiments are conducted in a test cell containing a cooled copper or brass plate with an exposed diameter of 12.7 cm. The metal surface is polished to allow for double-pass shadowgraph imaging, and the test surface is instrumented with imbedded heat transfer gauges and thermocouples. Representative shadowgraph images of a condensing, unstable (-1g) n-pentane film are shown. The interfacial disturbances associated with the de-stabilizing body force leading to droplet formation and break-off can be clearly seen. The heat transfer coefficient associated with the condensing film is shown. The heat transfer coefficient is seen to initially decrease, consistent with the increased thermal resistance due to layer growth. For sufficiently long time, a steady value of heat transfer is observed, accompanied by continuous droplet formation and break-off. The non-condensing cell consists of a stack of thin stainless steel disks 10 cm in diameter mounted in a brass enclosure. The disks are perforated with a regular pattern of 361 holes each 0.25 mm in diameter

  10. Viral mediated gene transfer to sprouting blood vessels during angiogenesis.

    PubMed

    Alian, Akram; Eldor, Amiram; Falk, Haya; Panet, Amos

    2002-08-01

    Several experimental systems have been applied to investigate the development of new blood vessels. Angiogenesis can be followed ex-vivo by culturing explants of rat aorta 'rings' in biomatrix gels. This angiogenesis system was modified for the study of viral vector mediated gene transfer, using adenovirus, vaccinia- and retroviral vectors. Two modifications were introduced to the model in order to facilitate efficient viral mediated gene transfer, (i) placing the aorta ring on top of a thin layer of collagen such that the angiogenic tissue will be accessible to the viral vector; and (ii) infection of the aorta rings prior to embedding them into the collagen matrix. While adenovirus and vaccinia vectors infected efficiently the aorta rings they induced cell death. Subsequent gene transfer experiments were, therefore, carried with retroviral vectors containing vascular endothelial growth factor (VEGF) and the beta-interferon (IFN) genes. Overexpression of VEGF enhanced significantly microvessel sprouting, while overexpression of IFN-beta induced an antiviral effect. The experimental system described in this study can facilitate the application of other viral vectors to the study of genes that may regulate the complex angiogenic process and thereby open new avenues for vascular gene therapy. PMID:12176137

  11. 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

  12. 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

  13. Detecting Horizontal Gene Transfer between Closely Related Taxa

    PubMed Central

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

    2015-01-01

    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

  14. 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

  15. 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...

  16. Transferred interbacterial antagonism genes augment eukaryotic innate immune function

    PubMed Central

    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.

    2015-01-01

    Horizontal gene transfer (HGT) allows organisms to rapidly acquire adaptive traits1. Though documented instances of HGT from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option2. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce anti-bacterials, 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 (T6SS)3. 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 via 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 etiologic 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 facile co-option by eukaryotic innate immune systems. PMID:25470067

  17. Detection of horizontal gene transfers from phylogenetic comparisons.

    PubMed

    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

  18. 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

  19. 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. The authors 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 x 10 /sup -4/-3 x 10/sup -3/. This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.

  20. AAV-mediated gene transfer to the mouse CNS

    PubMed Central

    Stoica, Lorelei; Ahmed, Seemin S.

    2013-01-01

    Recombinant adeno associated virus (rAAV) vectors are great tools for gene transfer due to their ability to mediate long-term gene expression. Recombinant AAVs have been used at various ages of development with no apparent toxicity. There are multiple ways of delivering AAV vectors to the CNS, depending on the stage of development of the mouse. In neonates, intravascular injections into the facial vein are often used. In adults, direct injections into target regions of the brain are achieved with great spatiotemporal control through stereotaxic surgeries. Recently, discoveries of new AAV vectors with the ability to cross the blood brain barrier have made it possible to also target the adult CNS by intravascular injections. rAAVs have been successfully used as gene transfer vehicles in multiple animal models of CNS disorders, and several clinical trials are currently underway. PMID:23686825

  1. 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

  2. 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

  3. Horizontal gene transfer confers adaptive advantages to phytopathogenic fungi: a case study of catalase-peroxidase in Fusarium verticillioides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Horizontal gene transfer (HGT), the exchange and stable integration of genetic material between different evolutionary lineages, is widely observed in fungi. We hypothesize that successful stabilization of HGT elements provides adaptive advantages (e.g., virulence). Catalase/peroxidases (KatGs) are ...

  4. 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.

  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. [Experimental study of vascular gene transfer using soluble stent].

    PubMed

    Huang, Z; Gou, J; Li, X

    1997-05-01

    We assessed the possibility of gene transfer into anastomotic arteries in vivo using soluble stent containing Adv5-CMV/LacZ. After being soaked in a high concentration solution of glucose containing Adv5-CMV (control group) or Adv5-CMV/LacZ (treatment group) for 30 minutes, we inserted soluble stents into the lumina of cut rat carotid arteries, and end-to-end anastomoses of cut rat carotid were performed with standard microvascular surgical technique. 16 rats were killed after two weeks, the segments of anastomotic carotid arteries were prepared for assessing beta-galactosidase activity and histochemical staining. In the control group, the anastomotic arteries did not have detectable level of beta-galactosidase expression. In the treatment group, the amount of beta-galactosidase expression was 9.80 x 10-3 u/g tissue. Microscopic examination of histochemically stained arteries demonstrated that gene transfered not only to endothelial cells but also to smooth muscle cells, and all anastomotic arteries were transfered in the treatment group, but none of arteries revealed blue in the control group. The results of this experimental study suggested that soluble stent be a new method of direct gene transfer into arteries in vivo. PMID:10374573

  7. Stability domains of actin genes and genomic evolution

    NASA Astrophysics Data System (ADS)

    Carlon, E.; Dkhissi, A.; Malki, M. Lejard; Blossey, R.

    2007-11-01

    In eukaryotic genes, the protein coding sequence is split into several fragments, the exons, separated by noncoding DNA stretches, the introns. Prokaryotes do not have introns in their genomes. We report calculations of the stability domains of actin genes for various organisms in the animal, plant, and fungi kingdoms. Actin genes have been chosen because they have been highly conserved during evolution. In these genes, all introns were removed so as to mimic ancient genes at the time of the early eukaryotic development, i.e., before intron insertion. Common stability boundaries are found in evolutionarily distant organisms, which implies that these boundaries date from the early origin of eukaryotes. In general, the boundaries correspond with intron positions in the actins of vertebrates and other animals, but not much for plants and fungi. The sharpest boundary is found in a locus where fungi, algae, and animals have introns in positions separated by one nucleotide only, which identifies a hot spot for insertion. These results suggest that some introns may have been incorporated into the genomes through a thermodynamically driven mechanism, in agreement with previous observations on human genes. They also suggest a different mechanism for intron insertion in plants and animals.

  8. Stability and functional effectiveness of phosphorothioate modified duplex DNA and synthetic 'mini-genes'.

    PubMed Central

    Ciafrè, S A; Rinaldi, M; Gasparini, P; Seripa, D; Bisceglia, L; Zelante, L; Farace, M G; Fazio, V M

    1995-01-01

    Several gene transfer techniques that employ 'naked DNA' molecules have recently been developed and numerous gene therapy protocols that make use of 'naked-DNA' have been proposed. We studied the possibility of enhancing the stability of 'naked DNA vectors' and thus also gene transfer and expression efficiencies, by constructing phosphorothioate (PS-) double strand DNA molecules and functional transcription units. We first synthesized short PS-double strand DNA molecules by the annealing of two complementary, 35 nt long, oligonucleotides. The accessibility of DNA modifying enzymes to this molecule was significantly decreased: T4-ligase and kinase activity were respectively reduced up to 1/2 and to 1/6, as compared to the normal phosphodiester molecule. Nucleolytic stability was increased either to purified enzymes (DNase I and Bal31) or to incubations in fresh serum, cell culture medium or in muscle protein extract. Phosphorothioate end-capped complete eukaryotic transcription units (obtained by Taq polymerase amplification with PS-primers) were not significantly protected from nucleolytic attack. On the contrary, synthetic transcription units, 'mini genes', obtained by Taq amplification with 1, 2 or 3 PS-dNTP substitutions, were resistant to DNase I and Bal31 nucleolytic activity. Transcription efficiency, driven by the T7 promoter, was 96.5, 95 and 33.5% (respectively with 1, 2 or 3 substitutions), as compared to the normal phosphodiester molecules. Images PMID:7479077

  9. Effect of radiative heat transfer on the convective stability of a fluid in a slot

    NASA Astrophysics Data System (ADS)

    Kural, O.

    1988-06-01

    A fluid, confined between two vertical flat plates, with a linear temperature gradient decreasing upwards, is investigated analytically for convective stability under the influence of radiative heat transfer. The effect of radiative transfer is accounted for by use of the Milne-Eddington differential approximation. It is shown that three dimensionless parameters influence the stability: the optical thickness, tau, a parameter A which compares radiative and conductive fluxes, and E, which combines the effects of boundary surface properties with the 'color' properties of the medium. It is shown that radiative heat transfer has a stabilizing effect on the system and that A and tau exert strong influences.

  10. Source–sink plasmid transfer dynamics maintain gene mobility in soil bacterial communities

    PubMed Central

    Wood, A. Jamie

    2016-01-01

    Horizontal gene transfer is a fundamental process in bacterial evolution that can accelerate adaptation via the sharing of genes between lineages. Conjugative plasmids are the principal genetic elements mediating the horizontal transfer of genes, both within and between bacterial species. In some species, plasmids are unstable and likely to be lost through purifying selection, but when alternative hosts are available, interspecific plasmid transfer could counteract this and maintain access to plasmid-borne genes. To investigate the evolutionary importance of alternative hosts to plasmid population dynamics in an ecologically relevant environment, we established simple soil microcosm communities comprising two species of common soil bacteria, Pseudomonas fluorescens and Pseudomonas putida, and a mercury resistance (HgR) plasmid, pQBR57, both with and without positive selection [i.e., addition of Hg(II)]. In single-species populations, plasmid stability varied between species: although pQBR57 survived both with and without positive selection in P. fluorescens, it was lost or replaced by nontransferable HgR captured to the chromosome in P. putida. A simple mathematical model suggests these differences were likely due to pQBR57’s lower intraspecific conjugation rate in P. putida. By contrast, in two-species communities, both models and experiments show that interspecific conjugation from P. fluorescens allowed pQBR57 to persist in P. putida via source–sink transfer dynamics. Moreover, the replacement of pQBR57 by nontransferable chromosomal HgR in P. putida was slowed in coculture. Interspecific transfer allows plasmid survival in host species unable to sustain the plasmid in monoculture, promoting community-wide access to the plasmid-borne accessory gene pool and thus potentiating future evolvability. PMID:27385827

  11. Source-sink plasmid transfer dynamics maintain gene mobility in soil bacterial communities.

    PubMed

    Hall, James P J; Wood, A Jamie; Harrison, Ellie; Brockhurst, Michael A

    2016-07-19

    Horizontal gene transfer is a fundamental process in bacterial evolution that can accelerate adaptation via the sharing of genes between lineages. Conjugative plasmids are the principal genetic elements mediating the horizontal transfer of genes, both within and between bacterial species. In some species, plasmids are unstable and likely to be lost through purifying selection, but when alternative hosts are available, interspecific plasmid transfer could counteract this and maintain access to plasmid-borne genes. To investigate the evolutionary importance of alternative hosts to plasmid population dynamics in an ecologically relevant environment, we established simple soil microcosm communities comprising two species of common soil bacteria, Pseudomonas fluorescens and Pseudomonas putida, and a mercury resistance (Hg(R)) plasmid, pQBR57, both with and without positive selection [i.e., addition of Hg(II)]. In single-species populations, plasmid stability varied between species: although pQBR57 survived both with and without positive selection in P. fluorescens, it was lost or replaced by nontransferable Hg(R) captured to the chromosome in P. putida A simple mathematical model suggests these differences were likely due to pQBR57's lower intraspecific conjugation rate in P. putida By contrast, in two-species communities, both models and experiments show that interspecific conjugation from P. fluorescens allowed pQBR57 to persist in P. putida via source-sink transfer dynamics. Moreover, the replacement of pQBR57 by nontransferable chromosomal Hg(R) in P. putida was slowed in coculture. Interspecific transfer allows plasmid survival in host species unable to sustain the plasmid in monoculture, promoting community-wide access to the plasmid-borne accessory gene pool and thus potentiating future evolvability. PMID:27385827

  12. Evaluation of Hose in Hose Transfer Line Service Life for Hanford's Interim Stabilization Program

    SciTech Connect

    TORRES, T.D.

    2000-08-24

    RPP-6153, Engineering Task Plan for Hose-in-Hose Transfer System for the Interim Stabilization Program, defines the programmatic goals, functional requirements, and technical criteria for the development and subsequent installation of transfer line equipment to support Hanford's Interim Stabilization Program. RPP-6028, Specification for Hose in Hose Transfer Lines for Hanford's Interim Stabilization Program, has been issued to define the specific requirements for the design, manufacture, and verification of transfer line assemblies for specific waste transfer applications. Included in RPP-6028 are tables defining the chemical constituents of concern to which transfer lines will be exposed. Current Interim Stabilization Program planning forecasts that the at-grade transfer lines will be required to convey pumpable waste for as much as three years after commissioning. Prudent engineering dictates that the equipment placed in service have a working life in excess of this forecasted time period, with some margin to allow for future adjustments to the planned schedule. This document evaluates the effective service life of the Hose-in-Hose Transfer Lines, based on information submitted by the manufacturer and published literature. The effective service life of transfer line assemblies is a function of several factors. Foremost among these are process fluid characteristics, ambient environmental conditions, and the manufacturer's stated shelf life. This evaluation examines the manufacturer's certification of shelf life, the manufacturer's certifications of chemical compatibility with waste, and published literature on the effects of exposure to ionizing radiation on the mechanical properties of elastomeric materials to evaluate transfer line service life.

  13. 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

  14. Electroporation-Mediated Gene Transfer Directly to the Swine Heart

    PubMed Central

    Hargrave, Barbara; Downey, Harre; Strange, Robert; Murray, Len; Cinnamond, Cade; Lundberg, Cathryn; Israel, Annelise; Chen, Yeong-Jer; Marshall, William; Heller, Richard

    2012-01-01

    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 3 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 ECG 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 euthanized 48 hours after injection and electroporation and gene expression was determined. Results were compared to 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 to 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. PMID:22456328

  15. 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

  16. 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

  17. A Rice Stowaway MITE for Gene Transfer in Yeast

    PubMed Central

    Fattash, Isam; Bhardwaj, Priyanka; Hui, Caleb; Yang, Guojun

    2013-01-01

    Miniature inverted repeat transposable elements (MITEs) lack protein coding capacity and often share very limited sequence similarity with potential autonomous elements. Their capability of efficient transposition and dramatic amplification led to the proposition that MITEs are an untapped rich source of materials for transposable element (TE) based genetic tools. To test the concept of using MITE sequence in gene transfer, a rice Stowaway MITE previously shown to excise efficiently in yeast was engineered to carry cargo genes (neo and gfp) for delivery into the budding yeast genome. Efficient excision of the cargo gene cassettes was observed even though the excision frequency generally decreases with the increase of the cargo sizes. Excised elements insert into new genomic loci efficiently, with about 65% of the obtained insertion sites located in genes. Elements at the primary insertion sites can be remobilized, frequently resulting in copy number increase of the element. Surprisingly, the orientation of a cargo gene (neo) on a construct bearing dual reporter genes (gfp and neo) was found to have a dramatic effect on transposition frequency. These results demonstrated the concept that MITE sequences can be useful in engineering genetic tools to deliver cargo genes into eukaryotic genomes. PMID:23704977

  18. Detection of homologous horizontal gene transfer in SNP data

    Energy Science and Technology Software Center (ESTSC)

    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.

  19. Stability of coaxial jets confined in a tube with heat and mass transfer

    NASA Astrophysics Data System (ADS)

    Mohanta, Lokanath; Cheung, Fan-Bill; Bajorek, Stephen M.

    2016-02-01

    A linear temporal stability of coaxial confined jets in a vertical tube involving heat and mass transfer at the interface is presented in this paper. A potential flow analysis that includes the effect of viscosity at the interface is performed in analyzing the stability of the system. Film boiling in a vertical tube gives rise to the flow configuration explored in this work. The effects of various non-dimensional parameters on the growth rate and the neutral curve are discussed. The heat transfer at the interface has been characterized by introducing a heat flux ratio between the conduction heat flux and the evaporation heat flux. Viscous forces and the heat and mass transfer at the interface are found to stabilize the flow both in the capillary instability region and Kelvin-Helmholtz instability region. Increasing heat and mass transfer at the interface stabilizes the flow to small as well as very large wave numbers.

  20. Probing the Mass-Transfer Stability Limit in Close Binaries

    NASA Astrophysics Data System (ADS)

    D'Souza, M. C. R.; Motl, P. M.; Tohline, J. E.; Frank, J.

    2004-12-01

    We present results from numerical simulations that follow the nonlinear development of mass-transfer instabilities in close binary star systems. A self-consistent-field (SCF) technique is used to construct initial equilibrium models, which are synchronously rotating, semi-detached, polytropic stars on circular orbits. These models are evolved with an Eulerian, finite-difference hydrodynamics code in a fully self-consistent manner. Results are presented for binary systems having a wide range of initial conditions; emphasis is placed on systems in which the mass-transfer stream directly impacts the surface of the accreting star. Some systems are dynamically unstable and result in a merger; others approach a long-term, stable phase of mass transfer. The time-evolutionary behavior of the binary separation, mass transfer rate, spin angular momentum of the accretor, and gravitational wave strain are presented.

  1. Horizontal Gene Transfer Regulation in Bacteria as a “Spandrel” of DNA Repair Mechanisms

    PubMed Central

    Fall, Saliou; Mercier, Anne; Bertolla, Franck; Calteau, Alexandra; Gueguen, Laurent; Perrière, Guy; Vogel, Timothy M.; Simonet, Pascal

    2007-01-01

    Horizontal gene transfer (HGT) is recognized as the major force for bacterial genome evolution. Yet, numerous questions remain about the transferred genes, their function, quantity and frequency. The extent to which genetic transformation by exogenous DNA has occurred over evolutionary time was initially addressed by an in silico approach using the complete genome sequence of the Ralstonia solanacearum GMI1000 strain. Methods based on phylogenetic reconstruction of prokaryote homologous genes families detected 151 genes (13.3%) of foreign origin in the R. solanacearum genome and tentatively identified their bacterial origin. These putative transfers were analyzed in comparison to experimental transformation tests involving 18 different genomic DNA positions in the genome as sites for homologous or homeologous recombination. Significant transformation frequency differences were observed among these positions tested regardless of the overall genomic divergence of the R. solanacearum strains tested as recipients. The genomic positions containing the putative exogenous DNA were not systematically transformed at the highest frequencies. The two genomic “hot spots”, which contain recA and mutS genes, exhibited transformation frequencies from 2 to more than 4 orders of magnitude higher than positions associated with other genes depending on the recipient strain. These results support the notion that the bacterial cell is equipped with active mechanisms to modulate acquisition of new DNA in different genomic positions. Bio-informatics study correlated recombination “hot-spots” to the presence of Chi-like signature sequences with which recombination might be preferentially initiated. The fundamental role of HGT is certainly not limited to the critical impact that the very rare foreign genes acquired mainly by chance can have on the bacterial adaptation potential. The frequency to which HGT with homologous and homeologous DNA happens in the environment might have led

  2. Selective Gene Transfer to the Retina Using Intravitreal Ultrasound Irradiation

    PubMed Central

    Sonoda, Shozo; Tachibana, Katsuro; Yamashita, Toshifumi; Shirasawa, Makoto; Terasaki, Hiroto; Uchino, Eisuke; Suzuki, Ryo; Maruyama, Kazuo; Sakamoto, Taiji

    2012-01-01

    This paper aims to evaluate the efficacy of intravitreal ultrasound (US) irradiation for green fluorescent protein (GFP) plasmid transfer into the rabbit retina using a miniature US transducer. Intravitreal US irradiation was performed by a slight modification of the transconjunctival sutureless vitrectomy system utilizing a small probe. After vitrectomy, the US probe was inserted through a scleral incision. A mixture of GFP plasmid (50 μL) and bubble liposomes (BLs; 50 μL) was injected into the vitreous cavity, and US was generated to the retina using a SonoPore 4000. The control group was not exposed to US. After 72 h, the gene-transfer efficiency was quantified by counting the number of GFP-positive cells. The retinas that received plasmid, BL, and US showed a significant increase in the number (average ± SEM) of GFP-positive cells (32 ± 4.9; n = 7; P < 0.01 ). No GFP-positive cells were observed in the control eyes (n = 7). Intravitreal retinal US irradiation can transfer the GFP plasmid into the retina without causing any apparent damage. This procedure could be used to transfer genes and drugs directly to the retina and therefore has potential therapeutic value. PMID:22518277

  3. 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.

  4. 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

  5. Evidence of horizontal gene transfer between obligate leaf nodule symbionts.

    PubMed

    Pinto-Carbó, Marta; Sieber, Simon; Dessein, Steven; Wicker, Thomas; Verstraete, Brecht; Gademann, Karl; Eberl, Leo; Carlier, Aurelien

    2016-09-01

    Bacteria of the genus Burkholderia establish an obligate symbiosis with plant species of the Rubiaceae and Primulaceae families. The bacteria, housed within the leaves, are transmitted hereditarily and have not yet been cultured. We have sequenced and compared the genomes of eight bacterial leaf nodule symbionts of the Rubiaceae plant family. All of the genomes exhibit features consistent with genome erosion. Genes potentially involved in the biosynthesis of kirkamide, an insecticidal C7N aminocyclitol, are conserved in most Rubiaceae symbionts. However, some have partially lost the kirkamide pathway due to genome erosion and are unable to synthesize the compound. Kirkamide synthesis is therefore not responsible for the obligate nature of the symbiosis. More importantly, we find evidence of intra-clade horizontal gene transfer (HGT) events affecting genes of the secondary metabolism. This indicates that substantial gene flow can occur at the early stages following host restriction in leaf nodule symbioses. We propose that host-switching events and plasmid conjugative transfers could have promoted these HGTs. This genomic analysis of leaf nodule symbionts gives, for the first time, new insights in the genome evolution of obligate symbionts in their early stages of the association with plants. PMID:26978165

  6. PEGylated Cationic Serum Albumin for Boosting Retroviral Gene Transfer.

    PubMed

    Palesch, David; Boldt, Felix; Müller, Janis A; Eisele, Klaus; Stürzel, Christina M; Wu, Yuzhou; Münch, Jan; Weil, Tanja

    2016-08-17

    Retroviral vectors are common tools for introducing genes into the genome of a cell. However, low transduction rates are a major limitation in retroviral gene transfer, especially in clinical applications. We generated cationic human serum albumin (cHSA) protected by a shell of poly(ethylene glycol) (PEG); this significantly enhanced retroviral gene transduction with potentially attractive pharmacokinetics and low immunogenicity. By screening a panel of chemically optimized HSA compounds, we identified a very potent enhancer that boosted the transduction rates of viral vectors. Confocal microscopy revealed a drastically increased number of viral particles attached to the surfaces of target cells. In accordance with the positive net charge of cationic and PEGylated HSA, this suggests a mechanism of action in which the repulsion of the negatively charged cellular and viral vector membranes is neutralized, thereby promoting attachment and ultimately transduction. Importantly, the transduction-enhancing PEGylated HSA derivative evaded recognition by HSA-specific antibodies and macrophage activation. Our findings hold great promise for facilitating improved retroviral gene transfer. PMID:27239020

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

    PubMed

    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

  8. 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

  9. PCR-based detection of gene transfer vectors: application to gene doping surveillance.

    PubMed

    Perez, Irene C; Le Guiner, Caroline; Ni, Weiyi; Lyles, Jennifer; Moullier, Philippe; Snyder, Richard O

    2013-12-01

    Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR. PMID:23912835

  10. Indications for Acquisition of Reductive Dehalogenase Genes through Horizontal Gene Transfer by Dehalococcoides ethenogenes Strain 195

    PubMed Central

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

    2005-01-01

    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

  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. Scapholunate stabilization with dynamic extensor carpi radialis longus tendon transfer.

    PubMed

    Peterson, Steven L; Freeland, Alan E

    2010-12-01

    Dynamic extensor carpi radialis longus tendon transfer to the distal pole of the scaphoid acts synchronously and synergistically with wrist motion to restore the slider crank mechanism of the scaphoid after scapholunate interosseous ligament (SLIL) injury. The procedure is designed to simulate a hypothetical dorsal radioscaphoid ligament that more closely approximates the normal viscoelastic forces acting on the scaphoid throughout all phases of wrist motion than does the static checkrein effect and motion limitations of capsulodesis or tenodesis. Extensor carpi radialis longus transfer may be independently sufficient to support normal or near-normal scapholunate and midcarpal kinematics and prevent further injury propagation in patients with partial SLIL tears and dynamic scapholunate instability. Extensor carpi radialis longus transfer alone may improve carpal congruity in patients with static scapholunate instability, but SLIL and dorsal lunate ligament repair or reconstruction is essential for favorable durable outcomes. Extensor carpi radialis longus transfer offers a simple and reasonable alternative to capsulodesis or tenodesis to support these ligament repairs or reconstructions, does not require intercarpal fixation, and allows rehabilitation to proceed expeditiously at approximately 1 month after surgery. PMID:21134618

  13. 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.

  14. 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. PMID:26412136

  15. 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

  16. 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.

  17. 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.

  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. PMID:26715630

  19. 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

  20. Thiolate versus Selenolate: Structure, Stability, and Charge Transfer Properties.

    PubMed

    Ossowski, Jakub; Wächter, Tobias; Silies, Laura; Kind, Martin; Noworolska, Agnieszka; Blobner, Florian; Gnatek, Dominika; Rysz, Jakub; Bolte, Michael; Feulner, Peter; Terfort, Andreas; Cyganik, Piotr; Zharnikov, Michael

    2015-04-28

    Selenolate is considered as an alternative to thiolate to serve as a headgroup mediating the formation of self-assembled monolayers (SAMs) on coinage metal substrates. There are, however, ongoing vivid discussions regarding the advantages and disadvantages of these anchor groups, regarding, in particular, the energetics of the headgroup-substrate interface and their efficiency in terms of charge transport/transfer. Here we introduce a well-defined model system of 6-cyanonaphthalene-2-thiolate and -selenolate SAMs on Au(111) to resolve these controversies. The exact structural arrangements in both types of SAMs are somewhat different, suggesting a better SAM-building ability in the case of selenolates. At the same time, both types of SAMs have similar packing densities and molecular orientations. This permitted reliable competitive exchange and ion-beam-induced desorption experiments which provided unequivocal evidence for a stronger bonding of selenolates to the substrate as compared to the thiolates. Regardless of this difference, the dynamic charge transfer properties of the thiolate- and selenolate-based adsorbates were found to be nearly identical, as determined by the core-hole-clock approach, which is explained by a redistribution of electron density along the molecular framework, compensating the difference in the substrate-headgroup bond strength. PMID:25857927

  1. Resistance Gene Transfer during Treatments for Experimental Avian Colibacillosis

    PubMed Central

    Dheilly, Alexandra; Le Devendec, Laëtitia; Mourand, Gwenaëlle; Bouder, Axelle; Jouy, Eric

    2012-01-01

    An experiment was conducted in animal facilities to compare the impacts of four avian colibacillosis treatments—oxytetracycline (OTC), trimethoprim-sulfadimethoxine (SXT), amoxicillin (AMX), or enrofloxacin (ENR)—on the susceptibility of Escherichia coli in broiler intestinal tracts. Birds were first orally inoculated with rifampin-resistant E. coli strains bearing plasmid genes conferring resistance to fluoroquinolones (qnr), cephalosporins (blaCTX-M or blaFOX), trimethoprim-sulfonamides, aminoglycosides, or tetracyclines. Feces samples were collected before, during, and after antimicrobial treatments. The susceptibilities of E. coli strains were studied, and resistance gene transfer was analyzed. An increase in the tetracycline-resistant E. coli population was observed only in OTC-treated birds, whereas multiresistant E. coli was detected in the dominant E. coli populations of SXT-, AMX-, or ENR-treated birds. Most multiresistant E. coli strains were susceptible to rifampin and exhibited various pulsed-field gel electrophoresis profiles, suggesting the transfer of one of the multiresistance plasmids from the inoculated strains to other E. coli strains in the intestinal tract. In conclusion, this study clearly illustrates how, in E. coli, “old” antimicrobials may coselect antimicrobial resistance to recent and critical molecules. PMID:21986830

  2. 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

  3. Lateral gene transfers have polished animal genomes: lessons from nematodes

    PubMed Central

    Danchin, Etienne G. J.; Rosso, Marie-Noëlle

    2012-01-01

    It is now accepted that lateral gene transfers (LGT), have significantly contributed to the composition of bacterial genomes. The amplitude of the phenomenon is considered so high in prokaryotes that it challenges the traditional view of a binary hierarchical tree of life to correctly represent the evolutionary history of species. Given the plethora of transfers between prokaryotes, it is currently impossible to infer the last common ancestral gene set for any extant species. For this ensemble of reasons, it has been proposed that the Darwinian binary tree of life may be inappropriate to correctly reflect the actual relations between species, at least in prokaryotes. In contrast, the contribution of LGT to the composition of animal genomes is less documented. In the light of recent analyses that reported series of LGT events in nematodes, we discuss the importance of this phenomenon in the evolutionary history and in the current composition of an animal genome. Far from being neutral, it appears that besides having contributed to nematode genome contents, LGT have favored the emergence of important traits such as plant-parasitism. PMID:22919619

  4. Stability analysis of electrical powered wheelchair-mounted robotic-assisted transfer device.

    PubMed

    Wang, Hongwu; Tsai, Chung-Ying; Jeannis, Hervens; Chung, Cheng-Shiu; Kelleher, Annmarie; Grindle, Garrett G; Cooper, Rory A

    2014-01-01

    The ability of people with disabilities to live in their homes and communities with maximal independence often hinges, at least in part, on their ability to transfer or be transferred by an assistant. Because of limited resources and the expense of personal care, robotic transfer assistance devices will likely be in great demand. An easy-to-use system for assisting with transfers, attachable to electrical powered wheelchairs (EPWs) and readily transportable, could have a significant positive effect on the quality of life of people with disabilities. We investigated the stability of our newly developed Strong Arm, which is attached and integrated with an EPW to assist with transfers. The stability of the system was analyzed and verified by experiments applying different loads and using different system configurations. The model predicted the distributions of the system's center of mass very well compared with the experimental results. When real transfers were conducted with 50 and 75 kg loads and an 83.25 kg dummy, the current Strong Arm could transfer all weights safely without tip-over. Our modeling accurately predicts the stability of the system and is suitable for developing better control algorithms to enhance the safety of the device. PMID:25356515

  5. 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

  6. Stability Analysis of Absorption Chiller-Heaters by Applying Transfer Function

    NASA Astrophysics Data System (ADS)

    Fujii, Tatsuo; Miyake, Satoshi; Oka, Masahiro; Mori, Kiyoyuki

    A transfer function approach is found to be a practical method for ensuring stable operation of absorption chiller-heaters. The transfer function model is based on a solution-circuit of the machine, which dominates the stability of the operation. This model includes a solution pump, a generator with an overflow weir, and a float valve. We found that the solution-circuit system is designed with the cascade control, which makes the system stable. In this construction, the float valve actuates a primary control loop, and the overflow weir actuates a secondary loop. The effects of the characteristic of the solution pump and the overflow weir are estimated by the degree of the stabilities, which are the gain margin and the phase margin. We found that the characteristic of the solution pump strongly effects the stability by enhancing the effect of the cascade control and improving the stability. So it is essential for a better stability analysis model. According to these results, the established model is useful for quantitatively predicting the stabilities of a chiller-heater in operation, and simultaneously reducing its size and improving the stability of operation. We conclude that the methodology based on transfer function can provide compact and reliable absorption chiller-heaters.

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

    PubMed

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

    2007-11-30

    Horizontal gene transfer, in which genetic material is transferred from the genome of one organism to that of 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 Escherichia 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. Our data suggest that toxicity to the host inhibited transfer regardless of the species of origin and that increased gene dosage and associated increased expression may be a predominant cause for transfer failure. Although these experimental studies examined transfer solely into E. coli, a computational analysis of gene-transfer rates across available bacterial and archaeal genomes supports that the barriers observed in our study are general across the tree of life. PMID:17947550

  8. 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.

  9. Repeated, recent and diverse transfers of a mitochondrial gene to the nucleus in flowering plants.

    PubMed

    Adams, K L; Daley, D O; Qiu, Y L; Whelan, J; Palmer, J D

    2000-11-16

    A central component of the endosymbiotic theory for the bacterial origin of the mitochondrion is that many of its genes were transferred to the nucleus. Most of this transfer occurred early in mitochondrial evolution; functional transfer of mitochondrial genes has ceased in animals. Although mitochondrial gene transfer continues to occur in plants, no comprehensive study of the frequency and timing of transfers during plant evolution has been conducted. Here we report frequent loss (26 times) and transfer to the nucleus of the mitochondrial gene rps10 among 277 diverse angiosperms. Characterization of nuclear rps10 genes from 16 out of 26 loss lineages implies that many independent, RNA-mediated rps10 transfers occurred during recent angiosperm evolution; each of the genes may represent a separate functional gene transfer. Thus, rps10 has been transferred to the nucleus at a surprisingly high rate during angiosperm evolution. The structures of several nuclear rps10 genes reveal diverse mechanisms by which transferred genes become activated, including parasitism of pre-existing nuclear genes for mitochondrial or cytoplasmic proteins, and activation without gain of a mitochondrial targeting sequence. PMID:11099041

  10. 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

  11. High-bandwidth transfer of phase stability through a fiber frequency comb.

    PubMed

    Scharnhorst, Nils; Wübbena, Jannes B; Hannig, Stephan; Jakobsen, Kornelius; Kramer, Johannes; Leroux, Ian D; Schmidt, Piet O

    2015-07-27

    We demonstrate phase locking of a 729 nm diode laser to a 1542 nm master laser via an erbium-doped-fiber frequency comb, using a transfer-oscillator feedforward scheme which suppresses the effect of comb noise in an unprecedented 1.8 MHz bandwidth. We illustrate its performance by carrying out coherent manipulations of a trapped calcium ion with 99 % fidelity even at few-μs timescales. We thus demonstrate that transfer-oscillator locking can provide sufficient phase stability for high-fidelity quantum logic manipulation even without pre-stabilization of the slave diode laser. PMID:26367634

  12. 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

  13. Gene stability in mammalian cells and protein consistency.

    PubMed

    Berthold, W

    1994-01-01

    The safety of a patient who is the recipient of protein drugs has to be assured. A "wrong" protein is thought to represent a great risk. The philosophy of testing strategies related to gene stability with product safety will be discussed in the light of experimental data available today. Although all mammalian cell lines used in the production of biologicals including recombinant DNA-derived lines have been produced from individual clones (functional monoclonality) they have been found to be heterogenous with regard to the genomic content (number of chromosomes, characteristics of identifiable chromosomes and position and number of integrated recombinant sequences). The verification of the presence of correct gene in a production cell line constitutes a well accepted and useful test, especially if derived by "population sequencing". A batch not related repeated confirmation of this fact cannot lead to any additional assurance for the correctness of all proteins constituting a given product beyond the level provided by cheminal testing. In contrast to this obvious and unavoidable heterogeneity in cellular genomes, the coding regions of genes have not been shown to change. Evidence is available to demonstrate the consistency of protein products originating from recombinant (and hybridoma) cell lines, e.g. more than 500,000 patients have received and tolerated rtPA well. PMID:7883100

  14. 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

  15. Comparison between Agrobacterium-mediated and direct gene transfer using the gene gun.

    PubMed

    Gao, Caixia; Nielsen, Klaus K

    2013-01-01

    Agrobacterium-mediated transformation and direct gene transfer using the gene gun (microparticle -bombardment) are the two most widely used methods for plant genetic modification. The Agrobacterium method has been successfully practiced in dicots for many years, but only recently have efficient protocols been developed for grasses. Microparticle bombardment has evolved as a method delivering exogenous nucleic acids into plant genome and is a commonly employed technique in plant science. Here these two systems are compared for transformation efficiency, transgene integration, and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The tall fescue transformation protocols lead to the production of large numbers of fertile, independent transgenic lines. PMID:23104329

  16. 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

  17. 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

  18. Phase transfer of oleic acid stabilized rod-shaped anatase TiO2 nanocrystals

    NASA Astrophysics Data System (ADS)

    Wilkerson, Rachel J.; Elder, Theresa; Sowinksi, Olivia; Fostvedt, Jade I.; Hoefelmeyer, James D.

    2016-06-01

    Three methods were evaluated for phase transfer of oleic acid stabilized TiO2 nanorods from non-polar phase to an aqueous phase. Three alkyltrimethylammonium bromide (C6, C8, C12) surfactants were tested and compared with an amphiphilic polymer as interdigitation agents. Ligand substitutions with catechol derivatives with polar functional groups para to the -enediol were evaluated as well. The molecular surfactants were ineffective compared to the amphiphilic polymer in the interdigitation phase transfer approach. Ligand substitution with catechols proceeded efficiently with phase transfer. The ligand substitution reactions were accompanied by gas evolution, which was found to result from decarboxylation of oleic acid in alkaline aqueous conditions.

  19. Stability-driven nonnegative matrix factorization to interpret spatial gene expression and build local gene networks.

    PubMed

    Wu, Siqi; Joseph, Antony; Hammonds, Ann S; Celniker, Susan E; Yu, Bin; Frise, Erwin

    2016-04-19

    Spatial gene expression patterns enable the detection of local covariability and are extremely useful for identifying local gene interactions during normal development. The abundance of spatial expression data in recent years has led to the modeling and analysis of regulatory networks. The inherent complexity of such data makes it a challenge to extract biological information. We developed staNMF, a method that combines a scalable implementation of nonnegative matrix factorization (NMF) with a new stability-driven model selection criterion. When applied to a set ofDrosophilaearly embryonic spatial gene expression images, one of the largest datasets of its kind, staNMF identified 21 principal patterns (PP). Providing a compact yet biologically interpretable representation ofDrosophilaexpression patterns, PP are comparable to a fate map generated experimentally by laser ablation and show exceptional promise as a data-driven alternative to manual annotations. Our analysis mapped genes to cell-fate programs and assigned putative biological roles to uncharacterized genes. Finally, we used the PP to generate local transcription factor regulatory networks. Spatially local correlation networks were constructed for six PP that span along the embryonic anterior-posterior axis. Using a two-tail 5% cutoff on correlation, we reproduced 10 of the 11 links in the well-studied gap gene network. The performance of PP with theDrosophiladata suggests that staNMF provides informative decompositions and constitutes a useful computational lens through which to extract biological insight from complex and often noisy gene expression data. PMID:27071099

  20. Improved gene transfer with histidine-functionalized mesoporous silica nanoparticles.

    PubMed

    Brevet, David; Hocine, Ouahiba; Delalande, Anthony; Raehm, Laurence; Charnay, Clarence; Midoux, Patrick; Durand, Jean-Olivier; Pichon, Chantal

    2014-08-25

    Mesoporous silica nanoparticles (MSN) were functionalized with aminopropyltriethoxysilane (MSN-NH2) then L-histidine (MSN-His) for pDNA delivery in cells and in vivo. The complexation of pDNA with MSN-NH2 and MSN-His was first studied with gel shift assay. pDNA complexed with MSN-His was better protected from DNase degradation than with MSN-NH2. An improvement of the transfection efficiency in cells was observed with MSN-His/pDNA compared to MSN-NH2/pDNA, which could be explained by a better internalization of MSN-His. The improvement of the transfection efficiency with MSN-His was also observed for gene transfer in Achilles tendons in vivo. PMID:24853464

  1. 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.

  2. Widespread impact of horizontal gene transfer on plant colonization of land

    PubMed Central

    Yue, Jipei; Hu, Xiangyang; Sun, Hang; Yang, Yongping; Huang, Jinling

    2012-01-01

    In complex multicellular eukaryotes such as animals and plants, horizontal gene transfer is commonly considered rare with very limited evolutionary significance. Here we show that horizontal gene transfer is a dynamic process occurring frequently in the early evolution of land plants. Our genome analyses of the moss Physcomitrella patens identified 57 families of nuclear genes that were acquired from prokaryotes, fungi or viruses. Many of these gene families were transferred to the ancestors of green or land plants. Available experimental evidence shows that these anciently acquired genes are involved in some essential or plant-specific activities such as xylem formation, plant defence, nitrogen recycling as well as the biosynthesis of starch, polyamines, hormones and glutathione. These findings suggest that horizontal gene transfer had a critical role in the transition of plants from aquatic to terrestrial environments. On the basis of these findings, we propose a model of horizontal gene transfer mechanism in nonvascular and seedless vascular plants. PMID:23093189

  3. 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. PMID:24432015

  4. 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

  5. Numerical Simulations of the Onset and Stability of Dynamical Mass Transfer in Binaries

    NASA Astrophysics Data System (ADS)

    D'Souza, Mario C. R.; Motl, Patrick M.; Tohline, Joel E.; Frank, Juhan

    2006-05-01

    Hydrodynamical simulations of semidetached, polytropic binary stars are presented in an effort to study the onset and stability of dynamical mass transfer events. Initial, synchronously rotating equilibrium models are constructed using a self-consistent field technique and then evolved with an Eulerian hydrodynamics code in a fully self-consistent manner. We describe code improvements introduced over the past few years that permit us to follow dynamical mass transfer events through more than 30 orbits. Mass transfer evolutions are presented for two different initial configurations: a dynamically unstable binary with initial mass ratio (donor/accretor) q0=1.3 that leads to a complete merger in ~10 orbits, and a double-degenerate binary with initial mass ratio q0=0.5 that, after some initial unstable growth of mass transfer, tends to separate as the mass transfer rate levels off.

  6. Multiple losses and transfers to the nucleus of two mitochondrial succinate dehydrogenase genes during angiosperm evolution.

    PubMed Central

    Adams, K L; Rosenblueth, M; Qiu, Y L; Palmer, J D

    2001-01-01

    Unlike in animals, the functional transfer of mitochondrial genes to the nucleus is an ongoing process in plants. All but one of the previously reported transfers in angiosperms involve ribosomal protein genes. Here we report frequent transfer of two respiratory genes, sdh3 and sdh4 (encoding subunits 3 and 4 of succinate dehydrogenase), and we also show that these genes are present and expressed in the mitochondria of diverse angiosperms. Southern hybridization surveys reveal that sdh3 and sdh4 have been lost from the mitochondrion about 40 and 19 times, respectively, among the 280 angiosperm genera examined. Transferred, functional copies of sdh3 and sdh4 were characterized from the nucleus in four and three angiosperm families, respectively. The mitochondrial targeting presequences of two sdh3 genes are derived from preexisting genes for anciently transferred mitochondrial proteins. On the basis of the unique presequences of the nuclear genes and the recent mitochondrial gene losses, we infer that each of the seven nuclear sdh3 and sdh4 genes was derived from a separate transfer to the nucleus. These results strengthen the hypothesis that angiosperms are experiencing a recent evolutionary surge of mitochondrial gene transfer to the nucleus and reveal that this surge includes certain respiratory genes in addition to ribosomal protein genes. PMID:11454775

  7. Kidney-specific Sonoporation-mediated Gene Transfer

    PubMed Central

    Ishida, Ryo; Kami, Daisuke; Kusaba, Tetsuro; Kirita, Yuhei; Kishida, Tsunao; Mazda, Osam; Adachi, Takaomi; Gojo, Satoshi

    2016-01-01

    Sonoporation can deliver agents to target local organs by systemic administration, while decreasing the associated risk of adverse effects. Sonoporation has been used for a variety of materials and in a variety of organs. Herein, we demonstrated that local sonoporation to the kidney can offer highly efficient transfer of oligonucleotides, which were systemically administrated to the tubular epithelium with high specificity. Ultrasonic wave irradiation to the kidney collapsed the microbubbles and transiently affected the glomerular filtration barrier and increased glomerular permeability. Oligonucleotides were passed through the barrier all at once and were absorbed throughout the tubular epithelium. Tumor necrosis factor alpha (TNFα), which plays a central role in renal ischemia–reperfusion injury, was targeted using small interfering RNA (siRNA) with renal sonoporation in a murine model. The reduction of TNFα expression after single gene transfer significantly inhibited the expression of kidney injury markers, suggesting that systemic administration of siRNA under temporary and local sonoporation could be applicable in the clinical setting of ischemic acute kidney injury. PMID:26419704

  8. Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases: An NHLBI Resource for the Gene Therapy Community

    PubMed Central

    Skarlatos, Sonia I.

    2012-01-01

    Abstract 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. PMID:22974119

  9. Engineering Task Plan for Hose In Hose Transfer Lines for the Interim Stabilization Program

    SciTech Connect

    RUNG, M.P.

    2000-06-20

    This document is the Engineering Task Plan for the engineering, design services, planning, project integration and management support for the design, modification, installation and testing of an over ground transfer (OGT) system to support the interim stabilization of nine tanks in the 241-S/SX Tank Farms.

  10. Engineering Task Plan for Hose In Hose Transfer Lines for the Interim Stabilization Program

    SciTech Connect

    TORRES, T.D.

    2000-08-16

    The document is the Engineering Task Plan for the engineering, design services, planning, project integration and management support for the design, modification, installation and testing of an over ground transfer (OGT) system to support the interim stabilization of S/SX and U Tank Farms.

  11. The tryptophanase gene cluster of Haemophilus influenzae type b: evidence for horizontal gene transfer.

    PubMed

    Martin, K; Morlin, G; Smith, A; Nordyke, A; Eisenstark, A; Golomb, M

    1998-01-01

    Among strains of Haemophilus influenzae, the ability to catabolize tryptophan (as detected by indole production) varies and is correlated with pathogenicity. Tryptophan catabolism is widespread (70 to 75%) among harmless respiratory isolates but is nearly universal (94 to 100%) among strains causing serious disease, including meningitis. As a first step in investigating the relationship between tryptophan catabolism and virulence, we have identified genes in pathogenic H. influenzae which are homologous to the tryptophanase (tna) operon of Escherichia coli. The tna genes are located on a 3.1-kb fragment between nlpD and mutS in the H. influenzae type b (Eagan) genome, are flanked by 43-bp direct repeats of an uptake signal sequence downstream from nlpD, and appear to have been inserted as a mobile unit within this sequence. The organization of this insertion is reminiscent of pathogenicity islands. The tna cluster is found at the same map location in all indole-positive strains of H. influenzae surveyed and is absent from reference type d and e genomes. In contrast to H. influenzae, most other Haemophilus species lack tna genes. Phylogenetic comparisons suggest that the tna cluster was acquired by intergeneric lateral transfer, either by H. influenzae or a recent ancestor, and that E. coli may have acquired its tnaA gene from a related source. Genomes of virulent H. influenzae resemble those of pathogenic enterics in having an island of laterally transferred DNA next to mutS. PMID:9422600

  12. Adenoviral-mediated gene transfer to rabbit synovium in vivo.

    PubMed Central

    Roessler, B J; Allen, E D; Wilson, J M; Hartman, J W; Davidson, B L

    1993-01-01

    Currently, treatment for rheumatoid arthritis and other inflammatory arthropathies is often ineffective in ameliorating the progression of the disease, particularly the invasive destruction of cartilage and bone by rheumatoid synovium. Multiple aspects of this inflammatory process are mediated by the synovial lining cells (synoviocytes). Genetic modification of these cells in vivo represents a potential method for the treatment of these conditions. In this report, we describe a novel technique for the genetic transduction of synovial lining cells in vivo using recombinant adenoviral vectors and intraarticular injection techniques. Purified high titer suspensions of a recombinant adenoviral vector containing the gene for Escherichia coli beta-galactosidase (AdCMVlacZ) were directly injected into the hind knees of New Zealand white rabbits. Synovial tissues were then examined for transgenic lacZ expression using a combination of in situ staining for beta-galactosidase activity, immunohistochemical staining, and transmission electron microscopy. High efficiency gene transfer and lacZ expression was observed in both type A and type B synoviocytes throughout the articular and periarticular synovium of the rabbit knee, with continued expression of transgenic lacZ detected for > or = 8 wk after infection. Images PMID:8349791

  13. Passive Immunization against HIV/AIDS by Antibody Gene Transfer

    PubMed Central

    Yang, Lili; Wang, Pin

    2014-01-01

    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. PMID:24473340

  14. 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.

  15. Horizontal transference of S-layer genes within Thermus thermophilus.

    PubMed Central

    Fernández-Herrero, L A; Olabarría, G; Castón, J R; Lasa, I; Berenguer, J

    1995-01-01

    The S-layers of Thermus thermophilus HB27 and T. thermophilus HB8 are composed of protein units of 95 kDa (P95) and 100 kDa (P100), respectively. We have selected S-layer deletion mutants from both strains by complete replacement of the slpA gene. Mutants of the two strains showed similar defects in growth and morphology and overproduced an external cell envelope inside of which cells remained after division. However, the nature of this external layer is strain specific, being easily stained and regular in the HB8 delta slpA derivative and amorphous and poorly stained in the HB27 delta slpA strain. The addition of chromosomic DNA from T. thermophilus HB8 to growing cultures of T. thermophilus HB27 delta slpA led to the selection of a new strain, HB27C8, which expressed a functional S-layer composed of the P100 protein. Conversely, the addition of chromosomic DNA from T. thermophilus HB27 to growing cultures of T. thermophilus HB8 delta slpA allowed the isolation of strain HB8C27, which expressed a functional S-layer composed of the P95 protein. The driving force which selected the transference of the S-layer genes in these experiments was the difference in growth rates, one of the main factors leading to selection in natural environments. PMID:7559330

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

    PubMed

    Yang, Lili; Wang, Pin

    2014-02-01

    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. PMID:24473340

  17. 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

  18. Frequent, independent transfers of a catabolic gene from bacteria to contrasted filamentous eukaryotes

    PubMed Central

    Bruto, Maxime; Prigent-Combaret, Claire; Luis, Patricia; Moënne-Loccoz, Yvan; Muller, Daniel

    2014-01-01

    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. PMID:24990676

  19. 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

  20. 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

  1. 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

  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. PMID:25733908

  3. Improved efficiency of the walnut somatic embryo gene transfer system.

    PubMed

    McGranahan, G H; Leslie, C A; Uratsu, S L; Dandekar, A M

    1990-01-01

    AnAgrobacterium-mediated gene transfer system which relies on repetitive embryogenesis to regenerate transgenic walnut plants has been made more efficient by using a more virulent strain ofAgrobacterium and vectors containing genes for both kanamycin resistance and beta-glucuronidase (GUS) activity to facilitate early screening and selection. Two plasmids (pCGN7001 and pCGN7314) introduced individually into the disarmedAgrobacterium host strain EHA101 were used as inoculum. Embryos maintained on medium containing 100 mg/l kanamycin after co-cultivation produced more transformed secondary embryos than embryos maintained on kanamycin-free medium. Of the 186 GUS-positive secondary embryo lines identified, 70% were regenerated from 3 out of 16 primary embryos inoculated with EHA101/pCGN7314 and grown on kanamycin- containing medium, 28% from 4 out of 17 primary embryos inoculated with EHA101/ pCGN7001 and grown on kanamycin medium, and 2% from one out of 13 primary embryos inoculated with EHA101/pCGN7001 but not exposed to kanamycin. Because kanamycin inhibits but does not completely block new embryo formation in controls, identification of transformants formerly required repetitive selection on kanamycin for several months. Introduction of the GUS marker gene allowed positive identification of transformant secondary embryos as early as 5-6 weeks after inoculation. DNA analysis of a representative subset of lines (n=13) derived from secondary embryos confirmed transformation and provided evidence for multiple insertion events in single inoculated primary embryos. PMID:24226275

  4. Total Transfer Capability Assessment Incorporating Corrective Controls for Transient Stability using TSCOPF

    NASA Astrophysics Data System (ADS)

    Hakim, Lukmanul; Kubokawa, Junji; Yorino, Naoto; Zoka, Yoshifumi; Sasaki, Yutaka

    Advancements have been made towards inclusion of both static and dynamic security into transfer capability calculation. However, to the authors' knowledge, work on considering corrective controls into the calculation has not been reported yet. Therefore, we propose a Total Transfer Capability (TTC) assessment considering transient stability corrective controls. The method is based on the Newton interior point method for nonlinear programming and transfer capability is approached as a maximization of power transfer with both static and transient stability constraints are incorporated into our Transient Stability Constrained Optimal Power Flow (TSCOPF) formulation. An interconnected power system is simulated to be subjected to a severe unbalanced 3-phase 4-line to ground fault and following the fault, generator and load are shed in a pre-defined sequence to mimic actual corrective controls. In a deregulated electricity market, both generator companies and large load customers are encouraged to actively participate in maintaining power system stability as corrective controls upon agreement of compensation for being shed following a disturbance. Implementation of this proposal on the actual power system operation should be carried out through combining it with the existing transient stabilization controller system. Utilization of these corrective controls results in increasing TTC as suggested in our numerical simulation. As Lagrange multipliers can also describe sensitivity of both inequality and equality constraints to the objective function, then selection of which generator or load to be shed can be carried out on the basis of values of Lagrange multipliers of its respective generator's rotor angle stability and active power balance equation. Hence, the proposal in this paper can be utilized by system operator to assess the maximum TTC for specific loads and network conditions.

  5. 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

  6. Persistent Gene Expression in Mouse Nasal Epithelia following Feline Immunodeficiency Virus-Based Vector Gene Transfer

    PubMed Central

    Sinn, Patrick L.; Burnight, Erin R.; Hickey, Melissa A.; Blissard, Gary W.; McCray, Paul B.

    2005-01-01

    Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 106 transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 107 to 109 TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (∼109 TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for ∼1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity. PMID:16188984

  7. Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish.

    PubMed

    Chenais, Nathalie; Lareyre, Jean-Jacques; Le Bail, Pierre-Yves; Labbe, Catherine

    2015-07-01

    The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression. Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. PMID:25929521

  8. Horizontal gene transfer of a Chlamydial tRNA-guanine transglycosylase gene to eukaryotic microbes.

    PubMed

    Manna, Sam; Harman, Ashley

    2016-01-01

    tRNA-guanine transglycosylases are found in all domains of life and mediate the base exchange of guanine with queuine in the anticodon loop of tRNAs. They can also regulate virulence in bacteria such as Shigella flexneri, which has prompted the development of drugs that inhibit the function of these enzymes. Here we report a group of tRNA-guanine transglycosylases in eukaryotic microbes (algae and protozoa) which are more similar to their bacterial counterparts than previously characterized eukaryotic tRNA-guanine transglycosylases. We provide evidence demonstrating that the genes encoding these enzymes were acquired by these eukaryotic lineages via horizontal gene transfer from the Chlamydiae group of bacteria. Given that the S. flexneri tRNA-guanine transglycosylase can be targeted by drugs, we propose that the bacterial-like tRNA-guanine transglycosylases could potentially be targeted in a similar fashion in pathogenic amoebae that possess these enzymes such as Acanthamoeba castellanii. This work also presents ancient prokaryote-to-eukaryote horizontal gene transfer events as an untapped resource of potential drug target identification in pathogenic eukaryotes. PMID:26435002

  9. Transduction-Like Gene Transfer in the Methanogen Methanococcus voltae

    PubMed Central

    Bertani, Giuseppe

    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 × 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. PMID:10321998

  10. 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.

  11. Self-assembled ternary complexes stabilized with hyaluronic acid-green tea catechin conjugates for targeted gene delivery.

    PubMed

    Liang, Kun; Bae, Ki Hyun; Lee, Fan; Xu, Keming; Chung, Joo Eun; Gao, Shu Jun; Kurisawa, Motoichi

    2016-03-28

    Nanosized polyelectrolyte complexes are attractive delivery vehicles for the transfer of therapeutic genes to diseased cells. Here we report the application of self-assembled ternary complexes constructed with plasmid DNA, branched polyethylenimine and hyaluronic acid-green tea catechin conjugates for targeted gene delivery. These conjugates not only stabilize plasmid DNA/polyethylenimine complexes via the strong DNA-binding affinity of green tea catechin, but also facilitate their transport into CD44-overexpressing cells via receptor-mediated endocytosis. The hydrodynamic size, surface charge and physical stability of the complexes are characterized. We demonstrate that the stabilized ternary complexes display enhanced resistance to nuclease attack and polyanion-induced dissociation. Moreover, the ternary complexes can efficiently transfect the difficult-to-transfect HCT-116 colon cancer cell line even in serum-supplemented media due to their enhanced stability and CD44-targeting ability. Confocal microscopic analysis demonstrates that the stabilized ternary complexes are able to promote the nuclear transport of plasmid DNA more effectively than binary complexes and hyaluronic acid-coated ternary complexes. The present study suggests that the ternary complexes stabilized with hyaluronic acid-green tea catechin conjugates can be widely utilized for CD44-targeted delivery of nucleic acid-based therapeutics. PMID:26855049

  12. Calfection: a novel gene transfer method for suspension cells.

    PubMed

    Lindell, Jeanette; Girard, Philippe; Müller, Natalie; Jordan, Martin; Wurm, Florian

    2004-01-20

    We have developed a novel method called Calfection for gene delivery to and protein expression from suspension-cultivated mammalian cells. Plasmid DNA was simply diluted into a calcium chloride solution and then added to the cell culture for transfection. We evaluated and optimized this approach using suspension-adapted HEK293 cells grown in 12-well plates that were shaken on an orbital shaker. Highest expression levels were obtained when cells were transfected at a density of 5x10(5) cells/ml in the presence of 9 mM calcium and 5 microg/ml of plasmid DNA while maintaining a culture pH of 7.6 at the time of transfection. Suspension-adapted BHK 21 and CHO DG 44 cells could also be transfected using this method. Calfection differs from the widely known calcium phosphate coprecipitation technique. The physico-chemical composition of the DNA interacting complexes is not yet known. The transfection cocktail, DNA in a calcium chloride solution, remained highly efficient during long-term storage at temperatures ranging from room temperature to -80 degrees C. In contrast, calcium phosphate-DNA cocktails are only efficient for gene transfer when prepared fresh. Furthermore, passing the calcium-plasmid DNA mixture through a 0.2-microm filter did not compromise protein expression, whereas calcium phosphate-DNA coprecipitates were retained by the filter. High protein expression levels, a limited number of manipulations and the possibility to filter the cocktail make the Calfection approach suitable for both large-scale transfection in bioreactors and for high-throughput transfection experiments in microtiter plates. PMID:14746910

  13. 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…

  14. 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

  15. Characterizing short-term stability for Boolean networks over any distribution of transfer functions

    NASA Astrophysics Data System (ADS)

    Seshadhri, C.; Smith, Andrew M.; Vorobeychik, Yevgeniy; Mayo, Jackson R.; Armstrong, Robert C.

    2016-07-01

    We present a characterization of short-term stability of Kauffman's N K (random) Boolean networks under arbitrary distributions of transfer functions. Given such a Boolean network where each transfer function is drawn from the same distribution, we present a formula that determines whether short-term chaos (damage spreading) will happen. Our main technical tool which enables the formal proof of this formula is the Fourier analysis of Boolean functions, which describes such functions as multilinear polynomials over the inputs. Numerical simulations on mixtures of threshold functions and nested canalyzing functions demonstrate the formula's correctness.

  16. Characterizing short-term stability for Boolean networks over any distribution of transfer functions.

    PubMed

    Seshadhri, C; Smith, Andrew M; Vorobeychik, Yevgeniy; Mayo, Jackson R; Armstrong, Robert C

    2016-07-01

    We present a characterization of short-term stability of Kauffman's NK (random) Boolean networks under arbitrary distributions of transfer functions. Given such a Boolean network where each transfer function is drawn from the same distribution, we present a formula that determines whether short-term chaos (damage spreading) will happen. Our main technical tool which enables the formal proof of this formula is the Fourier analysis of Boolean functions, which describes such functions as multilinear polynomials over the inputs. Numerical simulations on mixtures of threshold functions and nested canalyzing functions demonstrate the formula's correctness. PMID:27575142

  17. Gene transfer in the evolution of parasite nucleotide biosynthesis

    PubMed Central

    Striepen, Boris; Pruijssers, Andrea J. P.; Huang, Jinling; Li, Catherine; Gubbels, Marc-Jan; Umejiego, Nwakaso N.; Hedstrom, Lizbeth; Kissinger, Jessica C.

    2004-01-01

    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

  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. Neprilysin gene transfer: A promising therapeutic approach for Alzheimer's disease.

    PubMed

    Li, Yuanli; Wang, Junqing; Zhang, Shenghao; Liu, Zhaohui

    2015-09-01

    Alzheimer's disease (AD) is characterized by widespread neurodegeneration throughout the association cortex and limbic system, deposition of amyloid-β (Aβ) in the neuropil and around blood vessels, and formation of neurofibrillary tangles. Aβ accumulation is considered the major pathological change in AD progression. In recent years, several therapeutic strategies for treating AD have focused on reducing the Aβ burden in the brain. Among these approaches, the expression of Aβ-degrading enzymes in the brain has been effective but, so far, impractical for treating patients. Neprilysin (NEP), the most prominent of the Aβ-degrading enzymes in vivo, has been successfully delivered intracranially by viral vectors and is a promising therapeutic approach for reducing Aβ accumulation and treating AD. However, some challenges are associated with the use of viral and nonviral vectors, including secondary toxicity, activation of the immune response, and low efficiency. Therefore, safe and efficient NEP delivery systems that could avoid the viral problems with minor injury and high transfection efficiency are required to deliver AD medical applications. This Mini-Review summarizes NEP gene transfer technologies that use viral and nonviral vectors and discusses the rationale and benefits of these delivery systems for AD treatment trials, providing a reference for basic and clinical studies on AD. PMID:26096375

  20. Stagnation-point flow and heat transfer over an exponentially shrinking sheet: A stability analysis

    NASA Astrophysics Data System (ADS)

    Ismail, Nurul Syuhada; Arifin, Norihan Md.; Bachok, Norfifah; Mahiddin, Norhasimah

    2016-06-01

    Numerical solutions for the stagnation-point flow and heat transfer over an exponentially shrinking sheet have been investigated. The governing boundary layer equations are transformed into an ordinary differential equation using a non-similar transformation. By using the bvp4c solver in MATLAB, the results of the equations can be solved numerically. Numerical results indicate that in certain parameter, the non-unique solutions for the velocity and the temperature do exist. A linear stability analysis shows that only one solution is linearly stable otherwise is unstable. Then, the stability analysis is performed to identify which solution is stable between the two non-unique solutions.

  1. 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

  2. Phase transfer of citrate stabilized gold nanoparticles using nonspecifically adsorbed polymers.

    PubMed

    Alkilany, Alaaldin M; Caravana, Aidan C; Hamaly, Majd A; Lerner, Kevin T; Thompson, Lucas B

    2016-01-01

    Many synthetic approaches for gold nanoparticles rely on an aqueous media, resulting in water-soluble nanoparticles, which limits the ability to incorporate gold nanoparticles into other organic solvents or hydrophobic polymeric composites. Surface functionalization and phase transfer approaches using alkylthiols or alkylamines, which strongly bind the gold surface, are common routes to overcome this limitation, however they are typically challenging methods. In this paper we report an approach to transport citrate capped gold nanoparticles into a variety of solvents, including ones that are hydrophobic and not miscible with water without the need for phase transfer agents. We suspend gold nanoparticles in a water-miscible polar organic solvent that also is a solvent for a hydrophobic polymer. After drying, polymer-stabilized gold nanoparticles were found to be dispersible in various hydrophobic solvents with maintained colloidal stability. This work investigates two hydrophobic polymers, namely (polymethylmethacrylate and polyvinylacetate), which share common chemical motifs but have significantly different physiochemical properties. Interestingly, a significant difference in their ability to stabilize the transferred gold nanoparticles is observed and discussed. PMID:26397907

  3. Non-Viral Gene Transfer as a Tool for Studying Transcription Regulation of Xenobiotic Metabolizing Enzymes

    PubMed Central

    Bonamassa, Barbara; Liu, Dexi

    2010-01-01

    Numerous xenobiotic metabolizing enzymes are regulated by nuclear receptors at transcriptional level. The challenge we currently face is to understand how a given nuclear receptor interacts with its xenobiotics, migrates into nucleus, binds to the xenobiotic response element of a target gene, and regulates transcription. Toward this end, new methods have been developed to introduce the nuclear receptor gene into appropriate cells and study its activity in activating reporter gene expression under the control of a promoter containing xenobiotic response elements. The goal of this review is to critically examine the gene transfer methods currently available. We concentrate on the gene transfer mechanism, advantages and limitations of each method when employed for nuclear receptor-mediated gene regulation studies. It is our hope that the information provided highlights the importance of gene transfer in studying the mechanisms by which our body eliminates the potentially harmful substances and maintains the homeostasis. PMID:20713102

  4. Dendronization-induced phase-transfer, stabilization and self-assembly of large colloidal Au nanoparticles.

    PubMed

    Malassis, Ludivine; Jishkariani, Davit; Murray, Christopher B; Donnio, Bertrand

    2016-07-21

    The phase-transfer of CTAB-coated aqueous, spherical gold nanoparticles, with metallic core diameters ranging from ca. 27 to 54 nm, into organic solvents by exchanging the primitive polar bilayer with lipophilic, disulfide dendritic ligands is reported. The presence of such a thick nonpolar organic shell around these large nanoparticles enhances their stabilization against aggregation, in addition to enabling their transfer into a variety of solvents such as chloroform, toluene or tetrahydrofuran. Upon the slow evaporation of a chloroform suspension deposited on a solid support, the dendronized hybrids were found to self-assemble into ring structures of various diameters. Moreover, their self-assembly at the liquid-air interface affords the formation of fairly long-range ordered monolayers, over large areas, that can then be entirely transferred onto solid substrates. PMID:27348477

  5. 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

  6. Dendronization-induced phase-transfer, stabilization and self-assembly of large colloidal Au nanoparticles

    NASA Astrophysics Data System (ADS)

    Malassis, Ludivine; Jishkariani, Davit; Murray, Christopher B.; Donnio, Bertrand

    2016-07-01

    The phase-transfer of CTAB-coated aqueous, spherical gold nanoparticles, with metallic core diameters ranging from ca. 27 to 54 nm, into organic solvents by exchanging the primitive polar bilayer with lipophilic, disulfide dendritic ligands is reported. The presence of such a thick nonpolar organic shell around these large nanoparticles enhances their stabilization against aggregation, in addition to enabling their transfer into a variety of solvents such as chloroform, toluene or tetrahydrofuran. Upon the slow evaporation of a chloroform suspension deposited on a solid support, the dendronized hybrids were found to self-assemble into ring structures of various diameters. Moreover, their self-assembly at the liquid-air interface affords the formation of fairly long-range ordered monolayers, over large areas, that can then be entirely transferred onto solid substrates.The phase-transfer of CTAB-coated aqueous, spherical gold nanoparticles, with metallic core diameters ranging from ca. 27 to 54 nm, into organic solvents by exchanging the primitive polar bilayer with lipophilic, disulfide dendritic ligands is reported. The presence of such a thick nonpolar organic shell around these large nanoparticles enhances their stabilization against aggregation, in addition to enabling their transfer into a variety of solvents such as chloroform, toluene or tetrahydrofuran. Upon the slow evaporation of a chloroform suspension deposited on a solid support, the dendronized hybrids were found to self-assemble into ring structures of various diameters. Moreover, their self-assembly at the liquid-air interface affords the formation of fairly long-range ordered monolayers, over large areas, that can then be entirely transferred onto solid substrates. Electronic supplementary information (ESI) available: TEM microscope images. See DOI: 10.1039/c6nr03404g

  7. 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

  8. 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

  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. Genes under weaker stabilizing selection increase network evolvability and rapid regulatory adaptation to an environmental shift.

    PubMed

    Laarits, T; Bordalo, P; Lemos, B

    2016-08-01

    Regulatory networks play a central role in the modulation of gene expression, the control of cellular differentiation, and the emergence of complex phenotypes. Regulatory networks could constrain or facilitate evolutionary adaptation in gene expression levels. Here, we model the adaptation of regulatory networks and gene expression levels to a shift in the environment that alters the optimal expression level of a single gene. Our analyses show signatures of natural selection on regulatory networks that both constrain and facilitate rapid evolution of gene expression level towards new optima. The analyses are interpreted from the standpoint of neutral expectations and illustrate the challenge to making inferences about network adaptation. Furthermore, we examine the consequence of variable stabilizing selection across genes on the strength and direction of interactions in regulatory networks and in their subsequent adaptation. We observe that directional selection on a highly constrained gene previously under strong stabilizing selection was more efficient when the gene was embedded within a network of partners under relaxed stabilizing selection pressure. The observation leads to the expectation that evolutionarily resilient regulatory networks will contain optimal ratios of genes whose expression is under weak and strong stabilizing selection. Altogether, our results suggest that the variable strengths of stabilizing selection across genes within regulatory networks might itself contribute to the long-term adaptation of complex phenotypes. PMID:27213992

  11. 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

  12. 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

  13. 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.

  14. Proton Transfer to Flavin Stabilizes the Signaling State of the Blue Light Receptor Plant Cryptochrome*

    PubMed Central

    Hense, Anika; Herman, Elena; Oldemeyer, Sabine; Kottke, Tilman

    2015-01-01

    Plant cryptochromes regulate the circadian rhythm, flowering time, and photomorphogenesis in higher plants as responses to blue light. In the dark, these photoreceptors bind oxidized FAD in the photolyase homology region (PHR). Upon blue light absorption, FAD is converted to the neutral radical state, the likely signaling state, by electron transfer via a conserved tryptophan triad and proton transfer from a nearby aspartic acid. Here we demonstrate, by infrared and time-resolved UV-visible spectroscopy on the PHR domain, that replacement of the aspartic acid Asp-396 with cysteine prevents proton transfer. The lifetime of the radical is decreased by 6 orders of magnitude. This short lifetime does not permit to drive conformational changes in the C-terminal extension that have been associated with signal transduction. Only in the presence of ATP do both the wild type and mutant form a long-lived radical state. However, in the mutant, an anion radical is formed instead of the neutral radical, as found previously in animal type I cryptochromes. Infrared spectroscopic experiments demonstrate that the light-induced conformational changes of the PHR domain are conserved in the mutant despite the lack of proton transfer. These changes are not detected in the photoreduction of the non-photosensory d-amino acid oxidase to the anion radical. In conclusion, formation of the anion radical is sufficient to generate a protein response in plant cryptochromes. Moreover, the intrinsic proton transfer is required for stabilization of the signaling state in the absence of ATP. PMID:25471375

  15. Proton transfer to flavin stabilizes the signaling state of the blue light receptor plant cryptochrome.

    PubMed

    Hense, Anika; Herman, Elena; Oldemeyer, Sabine; Kottke, Tilman

    2015-01-16

    Plant cryptochromes regulate the circadian rhythm, flowering time, and photomorphogenesis in higher plants as responses to blue light. In the dark, these photoreceptors bind oxidized FAD in the photolyase homology region (PHR). Upon blue light absorption, FAD is converted to the neutral radical state, the likely signaling state, by electron transfer via a conserved tryptophan triad and proton transfer from a nearby aspartic acid. Here we demonstrate, by infrared and time-resolved UV-visible spectroscopy on the PHR domain, that replacement of the aspartic acid Asp-396 with cysteine prevents proton transfer. The lifetime of the radical is decreased by 6 orders of magnitude. This short lifetime does not permit to drive conformational changes in the C-terminal extension that have been associated with signal transduction. Only in the presence of ATP do both the wild type and mutant form a long-lived radical state. However, in the mutant, an anion radical is formed instead of the neutral radical, as found previously in animal type I cryptochromes. Infrared spectroscopic experiments demonstrate that the light-induced conformational changes of the PHR domain are conserved in the mutant despite the lack of proton transfer. These changes are not detected in the photoreduction of the non-photosensory d-amino acid oxidase to the anion radical. In conclusion, formation of the anion radical is sufficient to generate a protein response in plant cryptochromes. Moreover, the intrinsic proton transfer is required for stabilization of the signaling state in the absence of ATP. PMID:25471375

  16. Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus.

    PubMed

    Monier, Adam; Pagarete, António; de Vargas, Colomban; Allen, Michael J; Read, Betsy; Claverie, Jean-Michel; Ogata, Hiroyuki

    2009-08-01

    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

  17. 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

  18. 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

  19. Horizontally transferred genes in the genome of Pacific white shrimp, Litopenaeus vannamei

    PubMed Central

    2013-01-01

    Background In recent years, as the development of next-generation sequencing technology, a growing number of genes have been reported as being horizontally transferred from prokaryotes to eukaryotes, most of them involving arthropods. As a member of the phylum Arthropoda, the Pacific white shrimp Litopenaeus vannamei has to adapt to the complex water environments with various symbiotic or parasitic microorganisms, which provide a platform for horizontal gene transfer (HGT). Results In this study, we analyzed the genome-wide HGT events in L. vannamei. Through homology search and phylogenetic analysis, followed by experimental PCR confirmation, 14 genes with HGT event were identified: 12 of them were transferred from bacteria and two from fungi. Structure analysis of these genes showed that the introns of the two fungi-originated genes were substituted by shrimp DNA fragment, two genes transferred from bacteria had shrimp specific introns inserted in them. Furthermore, around other three bacteria-originated genes, there were three large DNA segments inserted into the shrimp genome. One segment was a transposon that fully transferred, and the other two segments contained only coding regions of bacteria. Functional prediction of these 14 genes showed that 6 of them might be related to energy metabolism, and 4 others related to defense of the organism. Conclusions HGT events from bacteria or fungi were happened in the genome of L. vannamei, and these horizontally transferred genes can be transcribed in shrimp. This is the first time to report the existence of horizontally transferred genes in shrimp. Importantly, most of these genes are exposed to a negative selection pressure and appeared to be functional. PMID:23914989

  20. 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.

  1. Nanoalumina promotes the horizontal transfer of multiresistance genes mediated by plasmids across genera

    PubMed Central

    Qiu, Zhigang; Yu, Yunmei; Chen, Zhaoli; Jin, Min; Yang, Dong; Zhao, Zuguo; Wang, Jingfeng; Shen, Zhiqiang; Wang, Xinwei; Qian, Di; Huang, Aihua; Zhang, Buchang; Li, Jun-Wen

    2012-01-01

    Antibiotic resistance is a worldwide public health concern. Conjugative transfer between closely related strains or species of bacteria is an important method for the horizontal transfer of multidrug-resistance genes. The extent to which nanomaterials are able to cause an increase in antibiotic resistance by the regulation of the conjugative transfer of antibiotic-resistance genes in bacteria, especially across genera, is still unknown. Here we show that nanomaterials in water can significantly promote the horizontal conjugative transfer of multidrug-resistance genes mediated by the RP4, RK2, and pCF10 plasmids. Nanoalumina can promote the conjugative transfer of the RP4 plasmid from Escherichia coli to Salmonella spp. by up to 200-fold compared with untreated cells. We also explored the mechanisms behind this phenomenon and demonstrate that nanoalumina is able to induce oxidative stress, damage bacterial cell membranes, enhance the expression of mating pair formation genes and DNA transfer and replication genes, and depress the expression of global regulatory genes that regulate the conjugative transfer of RP4. These findings are important in assessing the risk of nanomaterials to the environment, particularly from water and wastewater treatment systems, and in the estimation of the effect of manufacture and use of nanomaterials on the environment. PMID:22411796

  2. The Replication Checkpoint Protects Fork Stability by Releasing Transcribed Genes from Nuclear Pores

    PubMed Central

    Bermejo, Rodrigo; Capra, Thelma; Jossen, Rachel; Colosio, Arianna; Frattini, Camilla; Carotenuto, Walter; Cocito, Andrea; Doksani, Ylli; Klein, Hannah; Gómez-González, Belén; Aguilera, Andrés; Katou, Yuki; Shirahige, Katsuhiko; Foiani, Marco

    2011-01-01

    Summary Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes. PMID:21784245

  3. Lysogen stability is determined by the frequency of activity bursts from the fate-determining gene

    PubMed Central

    Zong, Chenghang; So, Lok-hang; Sepúlveda, Leonardo A; Skinner, Samuel O; Golding, Ido

    2010-01-01

    The ability of living cells to maintain an inheritable memory of their gene-expression state is key to cellular differentiation. Bacterial lysogeny serves as a simple paradigm for long-term cellular memory. In this study, we address the following question: in the absence of external perturbation, how long will a cell stay in the lysogenic state before spontaneously switching away from that state? We show by direct measurement that lysogen stability exhibits a simple exponential dependence on the frequency of activity bursts from the fate-determining gene, cI. We quantify these gene-activity bursts using single-molecule-resolution mRNA measurements in individual cells, analyzed using a stochastic mathematical model of the gene-network kinetics. The quantitative relation between stability and gene activity is independent of the fine details of gene regulation, suggesting that a quantitative prediction of cell-state stability may also be possible in more complex systems. PMID:21119634

  4. Lysogen stability is determined by the frequency of activity bursts from the fate-determining gene.

    PubMed

    Zong, Chenghang; So, Lok-hang; Sepúlveda, Leonardo A; Skinner, Samuel O; Golding, Ido

    2010-11-30

    The ability of living cells to maintain an inheritable memory of their gene-expression state is key to cellular differentiation. Bacterial lysogeny serves as a simple paradigm for long-term cellular memory. In this study, we address the following question: in the absence of external perturbation, how long will a cell stay in the lysogenic state before spontaneously switching away from that state? We show by direct measurement that lysogen stability exhibits a simple exponential dependence on the frequency of activity bursts from the fate-determining gene, cI. We quantify these gene-activity bursts using single-molecule-resolution mRNA measurements in individual cells, analyzed using a stochastic mathematical model of the gene-network kinetics. The quantitative relation between stability and gene activity is independent of the fine details of gene regulation, suggesting that a quantitative prediction of cell-state stability may also be possible in more complex systems. PMID:21119634

  5. Gene transfer into experimental brain tumors mediated by adenovirus, herpes simplex virus, and retrovirus vectors.

    PubMed

    Boviatsis, E J; Chase, M; Wei, M X; Tamiya, T; Hurford, R K; Kowall, N W; Tepper, R I; Breakefield, X O; Chiocca, E A

    1994-02-01

    Three vectors derived from retrovirus, herpes simplex virus type 1 (HSV), and adenovirus were compared in cultured rat 9L gliosarcoma cells for gene transfer efficiency and in a 9L rat brain tumor model for histologic pattern and distribution of foreign gene delivery, as well as for associated tumor necrosis and inflammation. At a multiplicity of infection of 1, in vitro transfer of a foreign gene (lacZ from Escherichia coli) into cells was more efficient with either the replication-defective retrovirus vector or the replication-conditional thymidine kinase (TK)-deficient HSV vector than with the replication-defective adenovirus vector. In vivo, stereotactic injections of each vector into rat brain tumors revealed three main histopathologic findings: (i) retrovirus and HSV vector-mediated gene transfer was relatively selective for cells within the tumor, whereas adenovirus vector-mediated gene transfer occurred into several types of endogenous neural cells, as well as into cells within the tumor; (ii) gene transfer to multiple infiltrating tumor deposits without apparent gene transfer to intervening normal brain tissue occurred uniquely in one animal inoculated with the HSV vector, and (iii) extensive necrosis and selective inflammation in the tumor were evident with the HSV vector, whereas there was minimal evidence of tumor necrosis and inflammation with either the retrovirus or adenovirus vectors. PMID:8186298

  6. A linear stability analysis for nonlinear, grey, thermal radiative transfer problems

    SciTech Connect

    Wollaber, Allan B.; Larsen, Edward W.

    2011-02-20

    We present a new linear stability analysis of three time discretizations and Monte Carlo interpretations of the nonlinear, grey thermal radiative transfer (TRT) equations: the widely used 'Implicit Monte Carlo' (IMC) equations, the Carter Forest (CF) equations, and the Ahrens-Larsen or 'Semi-Analog Monte Carlo' (SMC) equations. Using a spatial Fourier analysis of the 1-D Implicit Monte Carlo (IMC) equations that are linearized about an equilibrium solution, we show that the IMC equations are unconditionally stable (undamped perturbations do not exist) if {alpha}, the IMC time-discretization parameter, satisfies 0.5 < {alpha} {<=} 1. This is consistent with conventional wisdom. However, we also show that for sufficiently large time steps, unphysical damped oscillations can exist that correspond to the lowest-frequency Fourier modes. After numerically confirming this result, we develop a method to assess the stability of any time discretization of the 0-D, nonlinear, grey, thermal radiative transfer problem. Subsequent analyses of the CF and SMC methods then demonstrate that the CF method is unconditionally stable and monotonic, but the SMC method is conditionally stable and permits unphysical oscillatory solutions that can prevent it from reaching equilibrium. This stability theory provides new conditions on the time step to guarantee monotonicity of the IMC solution, although they are likely too conservative to be used in practice. Theoretical predictions are tested and confirmed with numerical experiments.

  7. 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

  8. Biomaterial-Mediated Retroviral Gene Transfer Using Self-Assembled Monolayers

    PubMed Central

    Gersbach, Charles A.; Coyer, Sean R.; Le Doux, Joseph M.; García, Andrés J.

    2007-01-01

    Biomaterial-mediated gene delivery has recently emerged as a promising alternative to conventional gene transfer technologies that focus on direct delivery of viral vectors or DNA-polymer/matrix complexes. However, biomaterial-based strategies have primarily targeted transient gene expression vehicles, including plasmid DNA and adenovirus particles. This study expands on this work by characterizing biomaterial properties conducive to the surface immobilization of retroviral particles and subsequent transduction of mammalian cells at the cell-material interface. Self-assembled monolayers (SAMs) of functionally-terminated alkanethiols on gold were used to establish biomaterial surfaces of defined chemical composition. Gene transfer was observed to be greater than 90% on NH2-terminated surfaces, approximately 50% on COOH-functionalized surfaces, and undetectable on CH3-terminated SAMs, similar to controls of tissue culture-treated polystyrene. Gene delivery via the NH2-SAM was further characterized as a function of coating time, virus concentration, and cell seeding density. Finally, SAM-mediated gene delivery was comparable to fibronectin- and poly-L-lysine-based methods for gene transfer. This work is significant to establishing safe and effective gene therapy strategies, developing efficient methods for gene delivery, and supporting recent progress in the field of biomaterial-mediated gene transfer. PMID:17698189

  9. Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish

    SciTech Connect

    Chenais, Nathalie; Lareyre, Jean-Jacques; Le Bail, Pierre-Yves; Labbe, Catherine

    2015-07-01

    The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression. Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. - Highlights: • Recycled fin explants outgrow cells bearing stable mesenchymal traits. • Cell production and quality is enhanced in the recycled explant culture system. • Fresh fin primary culture is highly variable and loose epithelial traits over time.

  10. An ancient horizontal gene transfer between mosquito and the endosymbiotic bacterium Wolbachia pipientis.

    PubMed

    Woolfit, Megan; Iturbe-Ormaetxe, Iñaki; McGraw, Elizabeth A; O'Neill, Scott L

    2009-02-01

    The extent and biological relevance of horizontal gene transfer (HGT) in eukaryotic evolution remain highly controversial. Recent studies have demonstrated frequent and large-scale HGT from endosymbiotic bacteria to their hosts, but the great majority of these transferred genes rapidly become nonfunctional in the recipient genome. Here, we investigate an ancient HGT between a host metazoan and an endosymbiotic bacterium, Wolbachia pipientis. The transferred gene has so far been found only in mosquitoes and Wolbachia. In mosquitoes, it is a member of a gene family encoding candidate receptors required for malaria sporozoite invasion of the mosquito salivary gland. The gene copy in Wolbachia has substantially diverged in sequence from the mosquito homolog, is evolving under purifying selection, and is expressed, suggesting that this gene is also functional in the bacterial genome. Several lines of evidence indicate that the gene may have been transferred from eukaryotic host to bacterial endosymbiont. Regardless of the direction of transfer, however, these results demonstrate that interdomain HGT may give rise to functional, persistent, and possibly evolutionarily significant new genes. PMID:18988686

  11. 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, ...

  12. An XMRV Derived Retroviral Vector as a Tool for Gene Transfer

    PubMed Central

    2011-01-01

    Background Retroviral vectors are widely used tools for gene delivery and gene therapy. They are useful for gene expression studies and genetic manipulation in vitro and in vivo. Many retroviral vectors are derived from the mouse gammaretrovirus, murine leukemia virus (MLV). These vectors have been widely used in gene therapy clinical trials. XMRV, initially found in prostate cancer tissue, was the first human gammaretrovirus described. Findings We developed a new retroviral vector based on XMRV called pXC. It was developed for gene transfer to human cells and is produced by transient cotransfection of LNCaP cells with pXC and XMRV-packaging plasmids. Conclusions We demonstrated that pXC mediates expression of inserted transgenes in cell lines. This new vector will be a useful tool for gene transfer in human and non-human cell lines, including gene therapy studies. PMID:21651801

  13. 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

  14. The Or gene enhances carotenoid accumulation and stability during post-harvest storage of potato tubers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Provitamin A carotenoids in staple crops are not very stable during storage and their loss compromises nutritional quality. To elucidate the fundamental mechanisms underlying carotenoid accumulation and stability, we investigated transgenic potato tubers that express the cauliflower Orange (Or) gene...

  15. Evaluation of Available Transfer Capability Using Transient Stability Constrained Line Flows

    NASA Astrophysics Data System (ADS)

    Uzoechi, Lazarus Okechukwu; Mahajan, Satish M.

    2014-01-01

    This paper presents a methodology to evaluate transient stability constrained available transfer capability (ATC). A linear and fast line flow-based (LFB) method was adopted to optimize the ATC values. This enabled the direct determination of the system source-sink locations. This paper formulated different market transactions considering bilateral and multilateral impacts in the stability constrained ATC. The proposed method was demonstrated on the WECC 9-bus and IEEE 39-bus systems. The critical energy performance index (CEPI) enabled the direct identification of candidates for contingency screening based on ranking. This index helped to reduce the list of credible contingencies for ATC evaluation and, therefore, the computation time. The results of the proposed ATC method are consistent with the literature and can be deployed for fast assessment of the impact of transactions in an electric power system.

  16. STABILITY EVALUATION OF METAL CASK ATTACHED TO A TRANSFER PALLET DURING LONG-PERIOD SEISMIC MOTIONS

    NASA Astrophysics Data System (ADS)

    Kawaguchi, Shohei; Shirai, Koji; Kanazawa, Kenji

    Rocking behavior of unfixed body is affected by center of mass, material coefficient of restitution and so on. 2/5 scale metal cask model considering these parameter was used for seismic test to evaluate stability of grounding metal cask attached to a transfer pallet under the influence of long-period earthquake motion. The newest knowledge from seismic test indicates seismic motion with high velocity over 100 kine not always cause the raise of response velocity of metal cask because of energy consumption by cask sliding and impact deformation of concrete. And new estimation method (called "Window energy spectrum method") of earthquake response spectrum gives suitable evaluation of response energy.

  17. Assessment of ICU readmission risk with the Stability and Workload Index for Transfer score*

    PubMed Central

    Oakes, Daiane Ferreira; Borges, Ingrid Nemitz Krás; Forgiarini, Luiz Alberto; Rieder, Marcelo de Mello

    2014-01-01

    Patient discharge from the ICU is indicated on the basis of clinical evidence and the result of strategies aimed at improving health care. Nevertheless, some patients might be discharged too early. We attempted to identify risk factors for unplanned ICU readmission, using a score for risk assessment, designated the Stability and Workload Index for Transfer (SWIFT) score. We evaluated 100 patients discharged from an ICU and found that the SWIFT score can be used as a tool for improving the assessment of ICU patients and the appropriateness of ICU discharge, thus preventing readmission. PMID:24626273

  18. 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

  19. Microarray analysis of relative gene expression stability for selection of internal reference genes in the rhesus macaque brain

    PubMed Central

    2010-01-01

    Background Normalization of gene expression data refers to the comparison of expression values using reference standards that are consistent across all conditions of an experiment. In PCR studies, genes designated as "housekeeping genes" have been used as internal reference genes under the assumption that their expression is stable and independent of experimental conditions. However, verification of this assumption is rarely performed. Here we assess the use of gene microarray analysis to facilitate selection of internal reference sequences with higher expression stability across experimental conditions than can be expected using traditional selection methods. We recently demonstrated that relative gene expression from qRT-PCR data normalized using GAPDH, ALG9 and RPL13A expression values mirrored relative expression using quantile normalization in Robust Multichip Analysis (RMA) on the Affymetrix® GeneChip® rhesus Macaque Genome Array. Having shown that qRT-PCR and Affymetrix® GeneChip® data from the same hormone replacement therapy (HRT) study yielded concordant results, we used quantile-normalized gene microarray data to identify the most stably expressed among probe sets for prospective internal reference genes across three brain regions from the HRT study and an additional study of normally menstruating rhesus macaques (cycle study). Gene selection was limited to 575 previously published human "housekeeping" genes. Twelve animals were used per study, and three brain regions were analyzed from each animal. Gene expression stabilities were determined using geNorm, NormFinder and BestKeeper software packages. Results Sequences co-annotated for ribosomal protein S27a (RPS27A), and ubiquitin were among the most stably expressed under all conditions and selection criteria used for both studies. Higher annotation quality on the human GeneChip® facilitated more targeted analysis than could be accomplished using the rhesus GeneChip®. In the cycle study, multiple

  20. Stability and toxicity of empty or gene-loaded lipopolysaccharide-amine nanopolymersomes

    PubMed Central

    Wang, Qinmei; Chen, Ying; Wang, Lichun; Zhang, Xinchun; Huang, Hongzhang; Teng, Wei

    2015-01-01

    Successful in vivo gene delivery mediated by nonviral vectors requires efficient extracellular and intracellular gene delivery, but few studies have given attention to the former. That is why numerous gene delivery systems have succeeded in vitro, while the expected clinical success has not come about. To realize efficient extracellular gene delivery, the stability of vectors and/or their complexes with genes in body fluids is first required, which prevents loaded genes from premature unloading and degradation. Furthermore, the storage stability of vectors under common conditions is important for their widespread applications. Lipopolysaccharide-amine nanopolymersomes (NPs), a gene vector developed by our group recently, have higher than 95% in vitro transfection efficiency in mesenchymal stem cells when delivering pEGFP, and induce significant angiogenesis in zebrafish when delivering plasmid encoding vascular endothelial growth factor deoxyribonucleic acid (pVEGF). To reveal their extracellular delivery ability and storage stability, in this study their stability in various simulant physiological environments and storage conditions was systematically studied by monitoring their changes in disassembly, size, zeta potential, and transfection efficiency. Additionally, damage to the mitochondria of mesenchymal stem cells was evaluated. Results show that NPs and plasmid deoxyribonucleic acid (pDNA)-loaded NPs (pNPs) have acceptable stability against dilution, anions, salts, pH, enzyme, and serum, presumably assuring their efficient extracellular delivery in vivo. Moreover, both the lyophilized NPs at room temperature and NP/pNP solution at 4°C have high storage stability, and pNPs show low damage to the mitochondria. The acceptable stability of NPs combined with compatibility and efficient gene transfection highlight their huge potential in the clinic as a gene delivery vector. PMID:25609964

  1. Development of second- and third-generation bovine immunodeficiency virus-based gene transfer systems.

    PubMed

    Matukonis, Meghan; Li, Mengtao; Molina, Rene P; Paszkiet, Brian; Kaleko, Michael; Luo, Tianci

    2002-07-20

    Lentivirus-based gene transfer systems have demonstrated their utility in mediating gene transfer to dividing and nondividing cells both in vitro and in vivo. An early-generation gene transfer system developed from bovine immunodeficiency virus (BIV) has been described (Berkowitz et al., J. Virol. 2001;75:3371-3382). In this paper, we describe the development of second-generation (three-plasmid) and third-generation (four-plasmid) BIV-based systems. All accessory genes (vif, vpw, vpy, and tmx) and the regulatory gene tat were deleted or largely truncated from the packaging construct. Furthermore, we split the packaging function into two constructs by expressing Rev in a separate plasmid. Together with our minimal BIV transfer vector construct and a vesicular stomatitis virus G glycoprotein-expressing plasmid, the BIV vectors were generated. The vectors produced by the three- and four-plasmid systems had titers greater than 1 x 10(6) transducing units per milliliter and were fully functional as indicated by their ability to efficiently transduce both dividing and nondividing cells. These results suggest that the accessory genes vif, vpw, vpy, and tmx are dispensable for functional BIV vector development. The modifications made to the packaging constructs improve the safety profile of the vector system. Finally, BIV vectors provide an alternative to human immunodeficiency virus-based gene transfer systems. PMID:12162812

  2. Exploration of horizontal gene transfer between transplastomic tobacco and plant-associated bacteria.

    PubMed

    Demanèche, Sandrine; Monier, Jean-Michel; Dugat-Bony, Eric; Simonet, Pascal

    2011-10-01

    The likelihood of gene transfer from transgenic plants to bacteria is dependent on the transgene copy number and on the presence of homologous sequences for recombination. The large number of chloroplast genomes in a plant cell as well as the prokaryotic origin of the transgene may thus significantly increase the likelihood of gene transfer from transplastomic plants to bacteria. In order to assess the probability of such a transfer, bacterial isolates, screened for their ability to colonize decaying tobacco plant tissue and possessing DNA sequence similarity to the chloroplastic genes accD and rbcL flanking the transgene (aadA), were tested for their ability to take up extracellular DNA (broad host-range pBBR1MCS-3-derived plasmid, transplastomic plant DNA and PCR products containing the genes accD-aadA-rbcL) by natural or electrotransformation. The results showed that among the 16 bacterial isolates tested, six were able to accept foreign DNA and acquire the spectinomycin resistance conferred by the aadA gene on plasmid, but none of them managed to integrate transgenic DNA in their chromosome. Our results provide no indication that the theoretical gene transfer-enhancing properties of transplastomic plants cause horizontal gene transfer at rates above those found in other studies with nuclear transgenes. PMID:21564143

  3. Chloroplast-like transfer RNA genes expressed in wheat mitochondria.

    PubMed Central

    Joyce, P B; Gray, M W

    1989-01-01

    In the course of a systematic survey of wheat mitochondrial tRNA genes, we have sequenced chloroplast-like serine (trnS-GGA), phenylalanine (trnF-GAA) and cysteine (trnC-GCA) tRNA genes and their flanking regions. These genes are remnants of 'promiscuous' chloroplast DNA that has been incorporated into wheat mtDNA in the course of its evolution. Each gene differs by one or a few nucleotides from the authentic chloroplast homolog previously characterized in wheat or other plants, and each could potentially encode a functional tRNA whose secondary structure shows no deviations from the generalized model. To determine whether these chloroplast-like tRNA genes are actually expressed, wheat mitochondrial tRNAs were resolved by a series of polyacrylamide gel electrophoreses, after being specifically end-labeled in vitro by 3'-CCA addition mediated by wheat tRNA nucleotidyltransferase. Subsequent direct RNA sequence analysis identified prominent tRNA species corresponding to the mitochondrial and not the chloroplast trnS, trnF and trnC genes. This analysis also revealed chloroplast-like elongator methionine, asparagine and tryptophan tRNAs. Our results suggest that at least some chloroplast-like tRNA genes in wheat mtDNA are transcribed, with transcripts undergoing processing, post-transcriptional modification and 3'-CCA addition, to produce mature tRNAs that may participate in mitochondrial protein synthesis. Images PMID:2762145

  4. Lentiviral vector-mediated gene transfer and RNA silencing technology in neuronal dysfunctions.

    PubMed

    Dreyer, Jean-Luc

    2011-02-01

    Lentiviral-mediated gene transfer in vivo or in cultured mammalian neurons can be used to address a wide variety of biological questions, to design animals models for specific neurodegenerative pathologies, or to test potential therapeutic approaches in a variety of brain disorders. Lentiviruses can infect non-dividing cells, thereby allowing stable gene transfer in post-mitotic cells such as mature neurons. An important contribution has been the use of inducible vectors: the same animal can thus be used repeatedly in the doxycycline-on or -off state, providing a powerful mean for assessing the function of a gene candidate in a disorder within a specific neuronal circuit. Furthermore, lentivirus vectors provide a unique tool to integrate siRNA expression constructs with the aim to locally knockdown expression of a specific gene, enabling to assess the function of a gene in a very specific neuronal pathway. Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in the brain. Therefore, the use of lentiviruses for stable expression of siRNA in brain is a powerful aid to probe gene functions in vivo and for gene therapy of diseases of the central nervous system. In this chapter I review the applications of lentivirus-mediated gene transfer in the investigation of specific gene candidates involved in major brain disorders and neurodegenerative processes. Major applications have been in polyglutamine disorders, such as synucleinopathies and Parkinson's disease, or in investigating gene function in Huntington's disease, dystonia, or muscular dystrophy. Recently, lentivirus gene transfer has been an invaluable tool for evaluation of gene function in behavioral disorders such as drug addiction and attention-deficit hyperactivity disorder or in learning and cognition. PMID:20862616

  5. Gene transfer into hematopoietic progenitor and stem cells: progress and problems.

    PubMed

    Dunbar, C E; Emmons, R V

    1994-11-01

    Gene transfer to hematopoietic cells for the purpose of "gene therapy" is a new and rapidly developing field with clinical trials in progress. A fundamental goal of research in this field is the incorporation of exogenous genes into the chromosomes of the most primitive hematopoietic progenitor cells--stem cells. Recombinantly engineered retroviral vectors are the best characterized and are currently the only vector type in clinical trials directed at the hematopoietic system. High efficiency gene transfer and expression in murine stem cells and their progeny is now routine, but in larger animal models such as dogs or primates and preliminary clinical trials, gene transfer has been less successful. Problems such as retroviral efficiency, gene expression, insertional mutagenesis and helper virus contamination are being addressed. A promising new vector, the adeno-associated virus (AAV), has shown promise and may allow production of high titer, stable, recombinant virions without helper contamination and with potentially better safety parameters. However, the technology for AAV gene transfer is currently underdeveloped, and issues related to the reproducible production of vectors must be addressed. Other non-viral vector systems are being explored, but little data are available on applications to hematopoietic cells. Better preclinical models are needed to study gene targeting and expression in human cells. An overview of recombinant retroviral and adeno-associated viral vector production, preclinical data and preliminary clinical data will be given, and problems needing to be addressed at all stages of development before broad clinical utility can be achieved will be discussed. PMID:7881358

  6. Evolutionary transfer of the chloroplast tufA gene to the nucleus.

    PubMed

    Baldauf, S L; Palmer, J D

    1990-03-15

    Evolutionary gene transfer is a basic corollary of the now widely accepted endosymbiotic theory, which proposes that mitochondria and chloroplasts originated from once free-living eubacteria. The small organellar chromosomes are remnants of larger bacterial genomes, with most endosymbiont genes having been either transferred to the nucleus soon after endosymbiosis or lost entirely, with some being functionally replaced by pre-existing nuclear genes. Several lines of evidence indicate that relocation of some organelle genes could have been more recent. These include the abundance of non-functional organelle sequences of recent origin in nuclear DNA, successful artificial transfer of functional organelle genes to the nucleus, and several examples of recently lost organelle genes, although none of these is known to have been replaced by a nuclear homologue that is clearly of organellar ancestry. We present gene sequence and molecular phylogenetic evidence for the transfer of the chloroplast tufA gene to the nucleus in the green algal ancestor of land plants. PMID:2314461

  7. The effect of asymmetric heating on flow stability and heat transfer for flow in a vertical tube

    SciTech Connect

    Tappan, C.H.

    1987-11-01

    This study presents experimental results of combined free and forced convection heat transfer in a vertical tube with a circumferentially nonuniform constant wall heat flux. The effect of an asymmetric wall heat flux on flow stability and on the rate of heat transfer for water flowing downward in a vertical tube was investigated. Experimental results were used to develop two stability maps which identify various flow regimes, corresponding to different thermal and hydraulic conditions. Heat transfer coefficients were also determined. Experimental results in the present investigation were compared to those with uniform heating in horizontal and vertical tube flow situations discussed in the literature. 23 refs., 12 figs., 1 tab.

  8. A unique horizontal gene transfer event has provided the octocoral mitochondrial genome with an active mismatch repair gene that has potential for an unusual self-contained function

    PubMed Central

    2011-01-01

    Background The mitochondrial genome of the Octocorallia has several characteristics atypical for metazoans, including a novel gene suggested to function in DNA repair. This mtMutS gene is favored for octocoral molecular systematics, due to its high information content. Several hypotheses concerning the origins of mtMutS have been proposed, and remain equivocal, although current weight of support is for a horizontal gene transfer from either an epsilonproteobacterium or a large DNA virus. Here we present new and compelling evidence on the evolutionary origin of mtMutS, and provide the very first data on its activity, functional capacity and stability within the octocoral mitochondrial genome. Results The mtMutS gene has the expected conserved amino acids, protein domains and predicted tertiary protein structure. Phylogenetic analysis indicates that mtMutS is not a member of the MSH family and therefore not of eukaryotic origin. MtMutS clusters closely with representatives of the MutS7 lineage; further support for this relationship derives from the sharing of a C-terminal endonuclease domain that confers a self-contained mismatch repair function. Gene expression analyses confirm that mtMutS is actively transcribed in octocorals. Rates of mitochondrial gene evolution in mtMutS-containing octocorals are lower than in their hexacoral sister-group, which lacks the gene, although paradoxically the mtMutS gene itself has higher rates of mutation than other octocoral mitochondrial genes. Conclusions The octocoral mtMutS gene is active and codes for a protein with all the necessary components for DNA mismatch repair. A lower rate of mitochondrial evolution, and the presence of a nicking endonuclease domain, both indirectly support a theory of self-sufficient DNA mismatch repair within the octocoral mitochondrion. The ancestral affinity of mtMutS to non-eukaryotic MutS7 provides compelling support for an origin by horizontal gene transfer. The immediate vector of transmission

  9. Horizontal Gene Transfers from Bacteria to Entamoeba Complex: A Strategy for Dating Events along Species Divergence

    PubMed Central

    Romero, Miguel; Ximenez, Cecilia

    2016-01-01

    Horizontal gene transfer has proved to be relevant in eukaryotic evolution, as it has been found more often than expected and related to adaptation to certain niches. A relatively large list of laterally transferred genes has been proposed and evaluated for the parasite Entamoeba histolytica. The goals of this work were to elucidate the importance of lateral gene transfer along the evolutionary history of some members of the genus Entamoeba, through identifying donor groups and estimating the divergence time of some of these events. In order to estimate the divergence time of some of the horizontal gene transfer events, the dating of some Entamoeba species was necessary, following an indirect dating strategy based on the fossil record of plausible hosts. The divergence between E. histolytica and E. nuttallii probably occurred 5.93 million years ago (Mya); this lineage diverged from E. dispar 9.97 Mya, while the ancestor of the latter separated from E. invadens 68.18 Mya. We estimated times for 22 transferences; the most recent occurred 31.45 Mya and the oldest 253.59 Mya. Indeed, the acquisition of genes through lateral transfer may have triggered a period of adaptive radiation, thus playing a major role in the evolution of the Entamoeba genus. PMID:27239333

  10. Horizontal Gene Transfers from Bacteria to Entamoeba Complex: A Strategy for Dating Events along Species Divergence.

    PubMed

    Romero, Miguel; Cerritos, R; Ximenez, Cecilia

    2016-01-01

    Horizontal gene transfer has proved to be relevant in eukaryotic evolution, as it has been found more often than expected and related to adaptation to certain niches. A relatively large list of laterally transferred genes has been proposed and evaluated for the parasite Entamoeba histolytica. The goals of this work were to elucidate the importance of lateral gene transfer along the evolutionary history of some members of the genus Entamoeba, through identifying donor groups and estimating the divergence time of some of these events. In order to estimate the divergence time of some of the horizontal gene transfer events, the dating of some Entamoeba species was necessary, following an indirect dating strategy based on the fossil record of plausible hosts. The divergence between E. histolytica and E. nuttallii probably occurred 5.93 million years ago (Mya); this lineage diverged from E. dispar 9.97 Mya, while the ancestor of the latter separated from E. invadens 68.18 Mya. We estimated times for 22 transferences; the most recent occurred 31.45 Mya and the oldest 253.59 Mya. Indeed, the acquisition of genes through lateral transfer may have triggered a period of adaptive radiation, thus playing a major role in the evolution of the Entamoeba genus. PMID:27239333

  11. Gene Transfers Shaped the Evolution of De Novo NAD+ Biosynthesis in Eukaryotes

    PubMed Central

    Ternes, Chad M.; Schönknecht, Gerald

    2014-01-01

    NAD+ is an essential molecule for life, present in each living cell. It can function as an electron carrier or cofactor in redox biochemistry and energetics, and serves as substrate to generate the secondary messenger cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate. Although de novo NAD+ biosynthesis is essential, different metabolic pathways exist in different eukaryotic clades. The kynurenine pathway starting with tryptophan was most likely present in the last common ancestor of all eukaryotes, and is active in fungi and animals. The aspartate pathway, detected in most photosynthetic eukaryotes, was probably acquired from the cyanobacterial endosymbiont that gave rise to chloroplasts. An evolutionary analysis of enzymes catalyzing de novo NAD+ biosynthesis resulted in evolutionary trees incongruent with established organismal phylogeny, indicating numerous gene transfers. Endosymbiotic gene transfers probably introduced the aspartate pathway into eukaryotes and may have distributed it among different photosynthetic clades. In addition, several horizontal gene transfers substituted eukaryotic genes with bacterial orthologs. Although horizontal gene transfer is accepted as a key mechanism in prokaryotic evolution, it is supposed to be rare in eukaryotic evolution. The essential metabolic pathway of de novo NAD+ biosynthesis in eukaryotes was shaped by numerous gene transfers. PMID:25169983

  12. Limitations of the murine nose in the development of nonviral airway gene transfer.

    PubMed

    Griesenbach, Uta; Sumner-Jones, Stephanie G; Holder, Emma; Munkonge, Felix M; Wodehouse, Theresa; Smith, Stephen N; Wasowicz, Marguerite Y; Pringle, Ian; Casamayor, Isabel; Chan, Mario; Coles, Rebecca; Cornish, Nikki; Dewar, Ann; Doherty, Ann; Farley, Raymond; Green, Anne-Marie; Jones, Bryony L; Larsen, Mia D B; Lawton, Anna E; Manvell, Michelle; Painter, Hazel; Singh, Charanjit; Somerton, Lucinda; Stevenson, Barbara; Varathalingam, Anusha; Siegel, Craig; Scheule, Ronald K; Cheng, Seng H; Davies, Jane C; Porteous, David J; Gill, Deborah R; Boyd, A Christopher; Hyde, Steve C; Alton, Eric W F W

    2010-07-01

    A clinical program to assess whether lipid GL67A-mediated gene transfer can ameliorate cystic fibrosis (CF) lung disease is currently being undertaken by the UK CF Gene Therapy Consortium. We have evaluated GL67A gene transfer to the murine nasal epithelium of wild-type and CF knockout mice to assess this tissue as a test site for gene transfer agents. The plasmids used were regulated by either (1) the commonly used short-acting cytomegalovirus promoter/enhancer or (2) the ubiquitin C promoter. In a study of approximately 400 mice with CF, vector-specific CF transmembrane conductance regulator (CFTR) mRNA was detected in nasal epithelial cells of 82% of mice treated with a cytomegalovirus-plasmid (pCF1-CFTR), and 62% of mice treated with an ubiquitin C-plasmid. We then assessed whether CFTR gene transfer corrected a panel of CFTR-specific endpoint assays in the murine nose, including ion transport, periciliary liquid height, and ex vivo bacterial adherence. Importantly, even with the comparatively large number of animals assessed, the CFTR function studies were only powered to detect changes of more than 50% toward wild-type values. Within this limitation, no significant correction of the CF phenotype was detected. At the current levels of gene transfer efficiency achievable with nonviral vectors, the murine nose is of limited value as a stepping stone to human trials. PMID:19648474

  13. Complexity of genetic sequences modified by horizontal gene transfer and degraded-DNA uptake

    NASA Astrophysics Data System (ADS)

    Tremberger, George; Dehipawala, S.; Nguyen, A.; Cheung, E.; Sullivan, R.; Holden, T.; Lieberman, D.; Cheung, T.

    2015-09-01

    Horizontal gene transfer has been a major vehicle for efficient transfer of genetic materials among living species and could be one of the sources for noncoding DNA incorporation into a genome. Our previous study of lnc- RNA sequence complexity in terms of fractal dimension and information entropy shows a tight regulation among the studied genes in numerous diseases. The role of sequence complexity in horizontal transferred genes was investigated with Mealybug in symbiotic relation with a 139K genome microbe and Deinococcus radiodurans as examples. The fractal dimension and entropy showed correlation R-sq of 0.82 (N = 6) for the studied Deinococcus radiodurans sequences. For comparison the Deinococcus radiodurans oxidative stress tolerant catalase and superoxide dismutase genes under extracellular dGMP growth condition showed R-sq ~ 0.42 (N = 6); and the studied arsenate reductase horizontal transferred genes for toxicity survival in several microorganisms showed no correlation. Simulation results showed that R-sq < 0.4 would be improbable at less than one percent chance, suggestive of additional selection pressure when compared to the R-sq ~ 0.29 (N = 21) in the studied transferred genes in Mealybug. The mild correlation of R-sq ~ 0.5 for fractal dimension versus transcription level in the studied Deinococcus radiodurans sequences upon extracellular dGMP growth condition would suggest that lower fractal dimension with less electron density fluctuation favors higher transcription level.

  14. Targeted gene transfer into rat facial muscles by nanosecond pulsed laser-induced stress waves

    NASA Astrophysics Data System (ADS)

    Kurita, Akihiro; Matsunobu, Takeshi; Satoh, Yasushi; Ando, Takahiro; Sato, Shunichi; Obara, Minoru; Shiotani, Akihiro

    2011-09-01

    We investigate the feasibility of using nanosecond pulsed laser-induced stress waves (LISWs) for gene transfer into rat facial muscles. LISWs are generated by irradiating a black natural rubber disk placed on the target tissue with nanosecond pulsed laser light from the second harmonics (532 nm) of a Q-switched Nd:YAG laser, which is widely used in head and neck surgery and proven to be safe. After injection of plasmid deoxyribose nucleic acid (DNA) coding for Lac Z into rat facial muscles, pulsed laser is used to irradiate the laser target on the skin surface without incision or exposure of muscles. Lac Z expression is detected by X-gal staining of excised rat facial skin and muscles. Strong Lac Z expression is observed seven days after gene transfer, and sustained for up to 14 days. Gene transfer is achieved in facial muscles several millimeters deep from the surface. Gene expression is localized to the tissue exposed to LISWs. No tissue damage from LISWs is observed. LISW is a promising nonviral target gene transfer method because of its high spatial controllability, easy applicability, and minimal invasiveness. Gene transfer using LISW to produce therapeutic proteins such as growth factors could be used to treat nerve injury and paralysis.

  15. Targeted gene transfer into rat facial muscles by nanosecond pulsed laser-induced stress waves.

    PubMed

    Kurita, Akihiro; Matsunobu, Takeshi; Satoh, Yasushi; Ando, Takahiro; Sato, Shunichi; Obara, Minoru; Shiotani, Akihiro

    2011-09-01

    We investigate the feasibility of using nanosecond pulsed laser-induced stress waves (LISWs) for gene transfer into rat facial muscles. LISWs are generated by irradiating a black natural rubber disk placed on the target tissue with nanosecond pulsed laser light from the second harmonics (532 nm) of a Q-switched Nd:YAG laser, which is widely used in head and neck surgery and proven to be safe. After injection of plasmid deoxyribose nucleic acid (DNA) coding for Lac Z into rat facial muscles, pulsed laser is used to irradiate the laser target on the skin surface without incision or exposure of muscles. Lac Z expression is detected by X-gal staining of excised rat facial skin and muscles. Strong Lac Z expression is observed seven days after gene transfer, and sustained for up to 14 days. Gene transfer is achieved in facial muscles several millimeters deep from the surface. Gene expression is localized to the tissue exposed to LISWs. No tissue damage from LISWs is observed. LISW is a promising nonviral target gene transfer method because of its high spatial controllability, easy applicability, and minimal invasiveness. Gene transfer using LISW to produce therapeutic proteins such as growth factors could be used to treat nerve injury and paralysis. PMID:21950944

  16. Synthetic gene transfer vectors II: back to the future.

    PubMed

    Behr, Jean-Paul

    2012-07-17

    The discovery of RNA interference has given a new lease on life to both the chemistry of oligonucleotides and chemical approaches for the intracellular delivery of nucleic acids. In particular, delivery of siRNA, whether in vitro for screening and target validation purposes or in humans as a new class of drugs, may revolutionize our approach to therapy. Their impact could equal that of the bioproduction and various uses of monoclonal antibodies today. Unfortunately, global pharmaceutical companies again seem to be waiting to buy the next Genentech or Genzyme of gene silencing rather than investing research and development into this promising area of research. Gene silencing encounters barriers similar to gene addition and hence may benefit from the extra decade of experience brought by gene therapy. "Chemical" transfection of cells in culture has become routine, and this Account discusses some of the reasons this success has not extended to nonviral gene therapy trials, most of which do not progress beyond the phase 2 stage. The author also discusses a (much debated) mechanism of nucleic acid cell entry and subsequent release of the polycationic particles into the cytoplasm. Both topics should be useful to those interested in delivery of siRNA. The move from gene therapy toward siRNA as an oligonucleotide-based therapy strategy provides a much wider range of druggable targets. Even though these molecules are a hundredfold smaller than a gene, they are delivered via similar cellular mechanisms. Their complexes with cationic polymers are less stable than those with a higher number of phosphate groups, which may be compensated by siRNA concatemerization or by chemical conjugation with the cationic carrier. Thus chemistry is again desperately needed. PMID:22311735

  17. Optical fiber-based photomechanical gene transfer system for in vivo application.

    PubMed

    Sato, Shunichi; Ando, Takahiro; Obara, Minoru

    2011-12-01

    We developed an optical-fiber-based photomechanical gene transfer system for endoscopic or catheter-based application. A fiber tip with a laser-absorbing film covered with a transparent plastic disk for plasma confinement was attached to a quartz fiber; the film was irradiated with nanosecond laser pulses transmitted through the fiber to generate photomechanical waves (PMWs). Characteristics of PMWs emitted from the fiber tip were examined to confirm the necessary conditions for gene transfer. We then attempted to transfer reporter genes to the rat skin as a test tissue in vivo with the fiber system, and the results showed significantly high protein levels and spatially selective pinpoint gene expressions in the tissue. PMID:22139237

  18. Activity-stability relationships revisited in blue oxidases catalyzing electron transfer at extreme temperatures.

    PubMed

    Roulling, Frédéric; Godin, Amandine; Cipolla, Alexandre; Collins, Tony; Miyazaki, Kentaro; Feller, Georges

    2016-09-01

    Cuproxidases are a subset of the blue multicopper oxidases that catalyze the oxidation of toxic Cu(I) ions into less harmful Cu(II) in the bacterial periplasm. Cuproxidases from psychrophilic, mesophilic, and thermophilic bacteria display the canonical features of temperature adaptation, such as increases in structural stability and apparent optimal temperature for activity with environmental temperature as well as increases in the binding affinity for catalytic and substrate copper ions. In contrast, the oxidative activities at 25 °C for both the psychrophilic and thermophilic enzymes are similar, suggesting that the nearly temperature-independent electron transfer rate does not require peculiar adjustments. Furthermore, the structural flexibilities of both the psychrophilic and thermophilic enzymes are also similar, indicating that the firm and precise bindings of the four catalytic copper ions are essential for the oxidase function. These results show that the requirements for enzymatic electron transfer, in the absence of the selective pressure of temperature on electron transfer rates, produce a specific adaptive pattern, which is distinct from that observed in enzymes possessing a well-defined active site and relying on conformational changes such as for the induced fit mechanism. PMID:27315165

  19. The agricultural antibiotic carbadox induces phage-mediated gene transfer in Salmonella

    PubMed Central

    Bearson, Bradley L.; Allen, Heather K.; Brunelle, Brian W.; Lee, In Soo; Casjens, Sherwood R.; Stanton, Thaddeus B.

    2013-01-01

    Antibiotics are used for disease therapeutic or preventative effects in humans and animals, as well as for enhanced feed conversion efficiency in livestock. Antibiotics can also cause undesirable effects in microbial populations, including selection for antibiotic resistance, enhanced pathogen invasion, and stimulation of horizontal gene transfer. Carbadox is a veterinary antibiotic used in the US during the starter phase of swine production for improved feed efficiency and control of swine dysentery and bacterial swine enteritis. Carbadox has been shown in vitro to induce phage-encoded Shiga toxin in Shiga toxin-producing Escherichia coli (STEC) and a phage-like element transferring antibiotic resistance genes in Brachyspira hyodysenteriae, but the effect of carbadox on prophages in other bacteria is unknown. This study examined carbadox exposure on prophage induction and genetic transfer in Salmonella enterica serovar Typhimurium, a human foodborne pathogen that frequently colonizes swine without causing disease. S. Typhimurium LT2 exposed to carbadox induced prophage production, resulting in bacterial cell lysis and release of virions that were visible by electron microscopy. Carbadox induction of phage-mediated gene transfer was confirmed by monitoring the transduction of a sodCIII::neo cassette in the Fels-1 prophage from LT2 to a recipient Salmonella strain. Furthermore, carbadox frequently induced generalized transducing phages in multidrug-resistant phage type DT104 and DT120 isolates, resulting in the transfer of chromosomal and plasmid DNA that included antibiotic resistance genes. Our research indicates that exposure of Salmonella to carbadox induces prophages that can transfer virulence and antibiotic resistance genes to susceptible bacterial hosts. Carbadox-induced, phage-mediated gene transfer could serve as a contributing factor in bacterial evolution during animal production, with prophages being a reservoir for bacterial fitness genes in the

  20. Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time.

    PubMed Central

    Zabner, J; Zeiher, B G; Friedman, E; Welsh, M J

    1996-01-01

    The efficiency of adenovirus-mediated gene transfer to airway epithelia will be an important factor in determining whether recombinant adenoviruses can be developed as vectors for transferring cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to patients with cystic fibrosis. Current understanding of the biology of CF lung disease suggests that vectors should express transgene in mature, ciliated airway epithelia. We evaluated the efficiency of adenovirus-mediated gene transfer to primary cultures of normal and CF human airway epithelia. Our studies showed that the airway cells developed from an undifferentiated epithelium with markers characteristic of basal cells and a surface covered by short microvilli 3 days after seeding to a mature epithelium whose apical surface was covered with cilia by 10 to 14 days. The ability of adenovirus vectors to express a reporter gene and to correct defective cyclic AMP-stimulated Cl- transport in CF epithelia was correlated inversely with the state of differentiation. However, the inefficiency of adenovirus-mediated gene transfer could be partially corrected when the contact time between vector and epithelium was prolonged. After prolonged contact, we observed complete correction of the CF Cl- transport defect in differentiated CF airway epithelia in culture and of the Cl- transport defect in the nasal epithelia of mice homozygous for the deltaF508 mutation. The fact that gene transfer to airway epithelia required prolonged incubation with vector contrasts with the rapid infection observed in cell models such as 293 and HeLa cells, which are commonly used to study adenovirus infection. Gene transfer observed after prolonged incubation may result from mechanisms different from those that mediate infection of 293 cells. These observations suggest that interventions that either increase the contact time or alter the epithelium or the vector may be required to facilitate gene transfer to ciliated respiratory epithelia

  1. Factors affecting stepladder stability during a lateral weight transfer: a study in healthy young adults.

    PubMed

    Yang, Bing-Shiang; Ashton-Miller, James A

    2005-09-01

    A fall from a stepladder is often initiated by a loss of lateral stability. An inverted pendulum model of the human, validated by experiment, was used to determine the feasible range of whole-body center of mass (COM) states for which weight can be transferred laterally on a ladder tread without a ladder rail losing contact with the ground ("no lift-off" stability region). The results show that the size of the feasible no lift-off region was inversely proportional to the height of the tread above the ground, the distance of the stance foot from the ipsilateral rail, and lateral ground inclination angle. For given initial COM kinematics on a tread height equal to 40% human body height, a stance-foot location equal to one-eighth tread width and a 3.5 degrees ground inclination had approximately equivalent effects on the no lift-off region size. Ladder stability was three times more sensitive to tread height than to foot location. Laterally-exerted impulsive hand-tool forces should generally be limited to 8% body weight. These findings can lead to improved ladder designs and safety instructions for stepladder users. PMID:15970205

  2. 4-HNE Adduct Stability Characterized by Collision-Induced Dissociation and Electron Transfer Dissociation Mass Spectrometry

    PubMed Central

    Fritz, Kristofer S.; Kellersberger, Katherine A.; Gomez, Jose D.; Petersen, Dennis R.

    2012-01-01

    4-hydroxynonenal (4-HNE) alters numerous proteomic and genomic processes. Understanding chemical mechanisms of 4-HNE interactions with biomolecules and their respective stabilities may lead to new discoveries in biomarkers for numerous diseases of oxidative stress. Collision-induced dissociation (CID) and electron transfer dissociation (ETD) MS/MS were utilized to examine the stability of a 4-HNE-Cys Michael adduct. CID conditions resulted in the neutral loss of 4-HNE, also known as a retro-Michael addition reaction (RMA). Consequently, performing ETD fragmentation on this same adduct did not result in RMA. Interestingly, 4-HNE adduct reduction via sodium borohydride (NaBH4) treatment stabilized against the CID induced RMA. In a direct comparison of three forms of 4-HNE adducts, computational modeling revealed sizeable shifts in the shape and orientation of the lowest unoccupied molecular orbital (LUMO) density around the 4-HNE-Cys moiety. These findings demonstrate that ETD MS/MS analysis can be used to improve the detection of 4-HNE-protein modifications by preventing RMA reactions from occurring. PMID:22404378

  3. Prokaryotic genes in eukaryotic genome sequences: when to infer horizontal gene transfer and when to suspect an actual microbe.

    PubMed

    Artamonova, Irena I; Lappi, Tanya; Zudina, Liudmila; Mushegian, Arcady R

    2015-07-01

    Assessment of phylogenetic positions of predicted gene and protein sequences is a routine step in any genome project, useful for validating the species' taxonomic position and for evaluating hypotheses about genome evolution and function. Several recent eukaryotic genome projects have reported multiple gene sequences that were much more similar to homologues in bacteria than to any eukaryotic sequence. In the spirit of the times, horizontal gene transfer from bacteria to eukaryotes has been invoked in some of these cases. Here, we show, using comparative sequence analysis, that some of those bacteria-like genes indeed appear likely to have been horizontally transferred from bacteria to eukaryotes. In other cases, however, the evidence strongly indicates that the eukaryotic DNA sequenced in the genome project contains a sample of non-integrated DNA from the actual bacteria, possibly providing a window into the host microbiome. Recent literature suggests also that common reagents, kits and laboratory equipment may be systematically contaminated with bacterial DNA, which appears to be sampled by metagenome projects non-specifically. We review several bioinformatic criteria that help to distinguish putative horizontal gene transfers from the admixture of genes from autonomously replicating bacteria in their hosts' genome databases or from the reagent contamination. PMID:25919787

  4. Formation of Stable Cationic Lipid/DNA Complexes for Gene Transfer

    NASA Astrophysics Data System (ADS)

    Hofland, Hans E. J.; Shephard, Lee; Sullivan, Sean M.

    1996-07-01

    Stable cationic lipid/DNA complexes were formed by solubilizing cationic liposomes with 1% octylglucoside and complexing a DNA plasmid with the lipid in the presence of detergent. Removal of the detergent by dialysis yielded a lipid/DNA suspension that was able to transfect tissue culture cells up to 90 days after formation with no loss in activity. Similar levels of gene transfer were obtained by mixing the cationic lipid in a liposome form with DNA just prior to cell addition. However, expression was completely lost 24 hr after mixing. The transfection efficiency of the stable complex in 15% fetal calf serum was 30% of that obtained in the absence of serum, whereas the transient complex was completely inactivated with 2% fetal calf serum. A 90-day stability study comparing various storage conditions showed that the stable complex could be stored frozen or as a suspension at 4 degrees C with no loss in transfection efficiency. Centrifugation of the stable complex produced a pellet that contained approximately 90% of the DNA and 10% of the lipid. Transfection of cells with the resuspended pellet and the supernatant showed that the majority of the transfection activity was in the pellet and all the toxicity was in the supernatant. Formation of a stable cationic lipid/DNA complex has produced a transfection vehicle that can be stored indefinitely, can be concentrated with no loss in transfection efficiency, and the toxicity levels can be greatly reduced when the active complex is isolated from the uncomplexed lipid.

  5. Two Horizontally Transferred Xenobiotic Resistance Gene Clusters Associated with Detoxification of Benzoxazolinones by Fusarium Species

    PubMed Central

    Glenn, Anthony E.; Davis, C. Britton; Gao, Minglu; Gold, Scott E.; Mitchell, Trevor R.; Proctor, Robert H.; Stewart, Jane E.; Snook, Maurice E.

    2016-01-01

    Microbes encounter a broad spectrum of antimicrobial compounds in their environments and often possess metabolic strategies to detoxify such xenobiotics. We have previously shown that Fusarium verticillioides, a fungal pathogen of maize known for its production of fumonisin mycotoxins, possesses two unlinked loci, FDB1 and FDB2, necessary for detoxification of antimicrobial compounds produced by maize, including the γ-lactam 2-benzoxazolinone (BOA). In support of these earlier studies, microarray analysis of F. verticillioides exposed to BOA identified the induction of multiple genes at FDB1 and FDB2, indicating the loci consist of gene clusters. One of the FDB1 cluster genes encoded a protein having domain homology to the metallo-β-lactamase (MBL) superfamily. Deletion of this gene (MBL1) rendered F. verticillioides incapable of metabolizing BOA and thus unable to grow on BOA-amended media. Deletion of other FDB1 cluster genes, in particular AMD1 and DLH1, did not affect BOA degradation. Phylogenetic analyses and topology testing of the FDB1 and FDB2 cluster genes suggested two horizontal transfer events among fungi, one being transfer of FDB1 from Fusarium to Colletotrichum, and the second being transfer of the FDB2 cluster from Fusarium to Aspergillus. Together, the results suggest that plant-derived xenobiotics have exerted evolutionary pressure on these fungi, leading to horizontal transfer of genes that enhance fitness or virulence. PMID:26808652

  6. CD133-targeted gene transfer into long-term repopulating hematopoietic stem cells.

    PubMed

    Brendel, Christian; Goebel, Benjamin; Daniela, Abriss; Brugman, Martijn; Kneissl, Sabrina; Schwäble, Joachim; Kaufmann, Kerstin B; Müller-Kuller, Uta; Kunkel, Hana; Chen-Wichmann, Linping; Abel, Tobias; Serve, Hubert; Bystrykh, Leonid; Buchholz, Christian J; Grez, Manuel

    2015-01-01

    Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells. PMID:25189742

  7. Carotenoids in unexpected places: gall midges, lateral gene transfer, and carotenoid biosynthesis in animals.

    PubMed

    Cobbs, Cassidy; Heath, Jeremy; Stireman, John O; Abbot, Patrick

    2013-08-01

    Carotenoids are conjugated isoprenoid molecules with many important physiological functions in organisms, including roles in photosynthesis, oxidative stress reduction, vision, diapause, photoperiodism, and immunity. Until recently, it was believed that only plants, microorganisms, and fungi were capable of synthesizing carotenoids and that animals acquired them from their diet, but recent studies have demonstrated that two arthropods (pea aphid and spider mite) possess a pair of genes homologous to those required for the first step of carotenoid biosynthesis. Absent in all other known animal genomes, these genes appear to have been acquired by aphids and spider mites in one or several lateral gene transfer events from a fungal donor. We report the third case of fungal carotenoid biosynthesis gene homologs in an arthropod: flies from the family Cecidomyiidae, commonly known as gall midges. Using phylogenetic analyses we show that it is unlikely that lycopene cyclase/phytoene synthase and phytoene desaturase homologs were transferred singly to an ancient arthropod ancestor; instead we propose that genes were transferred independently from related fungal donors after divergence of the major arthropod lineages. We also examine variation in intron placement and copy number of the carotenoid genes that may underlie function in the midges. This trans-kingdom transfer of carotenoid genes may represent a key innovation, underlying the evolution of phytophagy and plant-galling in gall midges and facilitating their extensive diversification across plant lineages. PMID:23542649

  8. Two Horizontally Transferred Xenobiotic Resistance Gene Clusters Associated with Detoxification of Benzoxazolinones by Fusarium Species.

    PubMed

    Glenn, Anthony E; Davis, C Britton; Gao, Minglu; Gold, Scott E; Mitchell, Trevor R; Proctor, Robert H; Stewart, Jane E; Snook, Maurice E

    2016-01-01

    Microbes encounter a broad spectrum of antimicrobial compounds in their environments and often possess metabolic strategies to detoxify such xenobiotics. We have previously shown that Fusarium verticillioides, a fungal pathogen of maize known for its production of fumonisin mycotoxins, possesses two unlinked loci, FDB1 and FDB2, necessary for detoxification of antimicrobial compounds produced by maize, including the γ-lactam 2-benzoxazolinone (BOA). In support of these earlier studies, microarray analysis of F. verticillioides exposed to BOA identified the induction of multiple genes at FDB1 and FDB2, indicating the loci consist of gene clusters. One of the FDB1 cluster genes encoded a protein having domain homology to the metallo-β-lactamase (MBL) superfamily. Deletion of this gene (MBL1) rendered F. verticillioides incapable of metabolizing BOA and thus unable to grow on BOA-amended media. Deletion of other FDB1 cluster genes, in particular AMD1 and DLH1, did not affect BOA degradation. Phylogenetic analyses and topology testing of the FDB1 and FDB2 cluster genes suggested two horizontal transfer events among fungi, one being transfer of FDB1 from Fusarium to Colletotrichum, and the second being transfer of the FDB2 cluster from Fusarium to Aspergillus. Together, the results suggest that plant-derived xenobiotics have exerted evolutionary pressure on these fungi, leading to horizontal transfer of genes that enhance fitness or virulence. PMID:26808652

  9. 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

  10. Horizontal transfer of archaeal genes into the deinococcaceae: detection by molecular and computer-based approaches

    NASA Technical Reports Server (NTRS)

    Olendzenski, L.; Liu, L.; Zhaxybayeva, O.; Murphey, R.; Shin, D. G.; Gogarten, J. P.

    2000-01-01

    Members of the Deinococcaceae (e.g., Thermus, Meiothermus, Deinococcus) contain A/V-ATPases typically found in Archaea or Eukaryotes which were probably acquired by horizontal gene transfer. Two methods were used to quantify the extent to which archaeal or eukaryotic genes have been acquired by this lineage. Screening of a Meiothermus ruber library with probes made against Thermoplasma acidophilum DNA yielded a number of clones which hybridized more strongly than background. One of these contained the prolyl tRNA synthetase (RS) gene. Phylogenetic analysis shows the M. ruber and D. radiodurans prolyl RS to be more closely related to archaeal and eukaryal forms of this gene than to the typical bacterial type. Using a bioinformatics approach, putative open reading frames (ORFs) from the prerelease version of the D. radiodurans genome were screened for genes more closely related to archaeal or eukaryotic genes. Putative ORFs were searched against representative genomes from each of the three domains using automated BLAST. ORFs showing the highest matches against archaeal and eukaryotic genes were collected and ranked. Among the top-ranked hits were the A/V-ATPase catalytic and noncatalytic subunits and the prolyl RS genes. Using phylogenetic methods, ORFs were analyzed and trees assessed for evidence of horizontal gene transfer. Of the 45 genes examined, 20 showed topologies in which D. radiodurans homologues clearly group with eukaryotic or archaeal homologues, and 17 additional trees were found to show probable evidence of horizontal gene transfer. Compared to the total number of ORFs in the genome, those that can be identified as having been acquired from Archaea or Eukaryotes are relatively few (approximately 1%), suggesting that interdomain transfer is rare.