Academic literature on the topic 'Prion protein gene'
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Journal articles on the topic "Prion protein gene"
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Scott, Michael R. D., Darel A. Butler, Dale E. Bredesen, Monika Wälchli, Karen K. Hsiao, and Stanley B. Prusiner. "Prion protein gene expression in cultured cells." "Protein Engineering, Design and Selection" 2, no. 1 (1988): 69–76. http://dx.doi.org/10.1093/protein/2.1.69.
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Nakamura, Yuko, Akikazu Sakudo, Keiichi Saeki, Tomomi Kaneko, Yoshitsugu Matsumoto, Antonio Toniolo, Shigeyoshi Itohara, and Takashi Onodera. "Transfection of prion protein gene suppresses coxsackievirus B3 replication in prion protein gene-deficient cells." Journal of General Virology 84, no. 12 (December 1, 2003): 3495–502. http://dx.doi.org/10.1099/vir.0.19222-0.
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The susceptibility of prion protein gene (Prnp)-null cells to coxsackievirus B3 (CVB3) was investigated. Primary cultures of murine Prnp −/− brain cells were more sensitive to CVBs than corresponding cells from wild-type mice. The viral susceptibility of a Prnp-null cell line (HpL3-4) derived from the murine hippocampus was compared with that of two established cell lines (HeLa and HEp-2) that are widely employed for CVB3 studies. After infection with CVB3, HpL3-4 cells showed a very rapid and complete cytopathic effect (CPE). CPE developed earlier and viruses replicated at higher titres in HpL3-4 cells compared with HeLa and HEp-2 cells. Under a semi-solid medium, plaques developed rapidly in CVB3-infected HpL3-4 cells. To confirm the effect of Prnp on virus infection, a Prnp −/− cell line and a Prnp-transfected neuronal cell line were analysed. The replication and release of infectious particles of CVB3 in Prnp −/− cells were significantly more effective than those of the Prnp-transfected cell line. Levels of type I interferon (IFN) after CVB3 infection were higher in the Prnp-transfected cell line than in Prnp −/− cells, whereas apoptotic cells were more obvious in the Prnp −/− cells than in those of the Prnp-transfected cell line. These findings suggest that the absence of Prnp retards the induction of CVB3-induced IFNs, resulting in an enhanced CVB3 production and apoptotic cell death. Furthermore, our data indicate that the HpL3-4 cell line may provide a novel and sensitive system for isolation of CVB3 from clinical specimens.
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Mironova, Ludmila N. "Protein inheritance and regulation of gene expression in yeast." Ecological genetics 8, no. 4 (December 15, 2010): 10–16. http://dx.doi.org/10.17816/ecogen8410-16.
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Prions of lower eukaryotes are genetic determinants of protein nature. Last years are marked by rapid development of the conception of prion inheritance. The list of yeast proteins, which have been shown to exist in the prion form in vivo, and phenotypic manifestation of prions provide good reason to believe that protein prionization may represent epigenetic mechanism regulating adaptability of a single cell and cellular population to environmental conditions.
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Caramelli, Paulo. "Prion protein gene in Alzheimer's disease." Arquivos de Neuro-Psiquiatria 71, no. 7 (July 2013): 419–20. http://dx.doi.org/10.1590/0004-282x20130093.
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Kovács, Gábor G., Gianriccardo Trabattoni, Johannes A. Hainfellner, James W. Ironside, Richard S. G. Knight, and Herbert Budka. "Mutations of the Prion Protein Gene." Journal of Neurology 249, no. 11 (November 1, 2002): 1567–82. http://dx.doi.org/10.1007/s00415-002-0896-9.
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Schätzl, Hermann M., Maria Da Costa, Leslie Taylor, Fred E. Cohen, and Stanley B. Prusiner. "Prion Protein Gene Variation Among Primates." Journal of Molecular Biology 245, no. 4 (January 1995): 362–74. http://dx.doi.org/10.1006/jmbi.1994.0030.
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Schätzl, Hermann M., Maria Da Costa, Leslie Taylor, Fred E. Cohen, and Stanley B. Prusiner. "Prion protein gene variation among primates." Journal of Molecular Biology 265, no. 2 (January 1997): 257. http://dx.doi.org/10.1006/jmbi.1996.0791.
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Mastrangelo, Peter, and David Westaway. "Biology of the prion gene complex." Biochemistry and Cell Biology 79, no. 5 (October 1, 2001): 613–28. http://dx.doi.org/10.1139/o01-142.
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The prion protein gene Prnp encodes PrPSc, the major structural component of prions, infectious pathogens causing a number of disorders including scrapie and bovine spongiform encephalopathy (BSE). Missense mutations in the human Prnp gene, PRNP, cause inherited prion diseases such as familial CreutzfeldtJakob Disease. In uninfected animals, Prnp encodes a GPI-anchored protein denoted PrPC, and in prion infections, PrPCis converted to PrPScby templated refolding. Although Prnp is conserved in mammalian species, attempts to verify interactions of putative PrP-binding proteins by genetic means have proven frustrating in that two independent lines of Prnp gene ablated mice (Prnp0/0mice: ZrchI and Npu) lacking PrPCremain healthy throughout development. This indicates that PrPCserves a function that is not apparent in a laboratory setting or that other molecules have overlapping functions. Shuttling or sequestration of synaptic Cu(II) via binding to N-terminal octapeptide residues and (or) signal transduction involving the fyn kinase are possibilities currently under consideration. A new point of entry into the issue of prion protein function has emerged from identification of a paralog, Prnd, with 25% coding sequence identity to Prnp. Prnd lies downstream of Prnp and encodes the Dpl protein. Like PrPC, Dpl is presented on the cell surface via a GPI anchor and has three α-helices: however, it lacks the conformationally plastic and octapeptide repeat domains present in its well-known relative. Interestingly, Dpl is overexpressed in two other lines of Prnp0/0mice (Ngsk and Rcm0) via intergenic splicing events. These lines of Prnp0/0mice exhibit ataxia and apoptosis of cerebellar cells, indicating that ectopic synthesis of Dpl protein is toxic to CNS neurons: this inference has now been confirmed by the construction of transgenic mice expressing Dpl under the direct control of the PrP promoter. Remarkably, Dpl-programmed ataxia is rescued by wt Prnp transgenes. The interaction between the Prnp and Prnd genes in mouse cerebellar neurons may have a physical correlate in competition between Dpl and PrPCwithin a common biochemical pathway that, when misregulated, leads to apoptosis.Key words: spongiform encephalopathy, neurodegenerative disease, paralogs, scrapie, CJD.
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Cardinale, Alessio, and Silvia Biocca. "Gene-Based Antibody Strategies for Prion Diseases." International Journal of Cell Biology 2013 (2013): 1–6. http://dx.doi.org/10.1155/2013/710406.
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Prion diseases or transmissible spongiform encephalopathies (TSE) are a group of neurodegenerative and infectious disorders characterized by the conversion of a normal cellular protein PrPCinto a pathological abnormally folded form, termed PrPSc. There are neither available therapies nor diagnostic tools for an early identification of individuals affected by these diseases. New gene-based antibody strategies are emerging as valuable therapeutic tools. Among these, intrabodies are chimeric molecules composed by recombinant antibody fragments fused to intracellular trafficking sequences, aimed at inhibiting,in vivo, the function of specific therapeutic targets. The advantage of intrabodies is that they can be selected against a precise epitope of target proteins, including protein-protein interaction sites and cytotoxic conformers (i.e., oligomeric and fibrillar assemblies). Herein, we address and discussin vitroandin vivoapplications of intrabodies in prion diseases, focussing on their therapeutic potential.
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Resende, Catarina G., Tiago F. Outeiro, Laina Sands, Susan Lindquist, and Mick F. Tuite. "Prion protein gene polymorphisms in Saccharomyces cerevisiae." Molecular Microbiology 49, no. 4 (July 4, 2003): 1005–17. http://dx.doi.org/10.1046/j.1365-2958.2003.03608.x.
Full textDissertations / Theses on the topic "Prion protein gene"
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Premzl, Marko, and Premzl@anu edu au premzl@excite com Marko. "Prion Protein Gene and Its Shadow." The Australian National University. The John Curtin School of Medical Research, 2004. http://thesis.anu.edu.au./public/adt-ANU20050328.164529.
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Prion protein (PrP) is best known for its involvement in prion diseases. A normal, dynamic isoform of prion protein (PrP^C) transforms into a pathogenic, compact isoform (PrP^Sc) during prion disease pathogenesis. The PrP^Sc, acting as a template upon which PrP^C molecules are refolded into a likeness of itself, accumulates in the brain neurones and causes disease. It is the only known component of prions, proteinaceous infectious particles. Both prion protein isoforms have the same primary amino acid structure and are encoded by the same prion protein gene (PRNP). PRNP determines susceptibility/disposition to prion diseases and their phenotypes.¶The normal function of PRNP is elusive. The Prnp knock-out mice with disrupted ORF show only very subtle phenotype. A number of hypotheses were proposed on the function of mammalian PRNP. The extracellular, GPI-anchored, glycosylated mammalian PrP^C expressed in a heterogenous set of cells could: transport copper from extracellular to intracellular milieu, buffer copper from synapse, contribute to redox signalling, act neuroprotectively, mediate cell-cell contacts, affect lymphocyte activation, participate in nucleic acid metabolism, be a memory molecule, and be a signal-transduction protein.¶ Experimental evidence demonstrated a redundancy between the PRNP and another, unknown gene. The critical issue therefore is to discover new genes homologous with PRNP, candidates for this redundancy. Using unpublished data, a sequence of zebrafish cDNA sequenced by Prof. Tatjana Simonics group (University of Milan, Italy), I discovered a new paralogue of PRNP. By searching manually, and in a targeted fashion, data deposited in public biological databases, I compiled support for the new human gene Shadow of prion protein (SPRN) including the direct evidence, homology-based evidence and ab initio gene prediction. The protein product called Shadoo (shadow in Japanese) is an extracellular, potentially glycosylated and GPI-anchored protein of a mature size of 100-odd amino acids. It is conserved from fish (zebrafish, Fugu, Tetraodon) to mammals (human, mouse, rat), and exhibits similarity of overall protein features with PrP. Most remarkably, the Sho is the first human/mammalian protein apart from PrP that contains the middle hydrophobic region that is essential for both normal and pathogenic properties of PrP. As this region is critical for heterodimerization of PrP, Sho may have potential to interact with PrP and is a likely candidate for the Protein X. Mammalian SPRN could be predominantly expressed in brain (Tatjana Simonic Lab, University of Milan, Italy).¶ Using the same approach to search public databases, I found, in addition, a fish duplicate of SPRN called SPRNB, and defined a new vertebrate SPRN gene family. Further, I also expanded a number of known fish genes from the PRNP gene family. The total number of the new genes that I discovered is 11. With the representatives of two vertebrate gene family datasets in hand, I conducted comparative genomic analysis in order to determine evolutionary trajectories of the SPRN and PRNP genes. This analysis, complemented with phylogenetic studies (Dr. Lars Jermiin, University of Sydney, Australia), demonstrated conservative evolution of the mammalian SPRN gene, and more relaxed evolutionary constraints acting on the mammalian PRNP gene. This evolutionary dialectic challenges widely adopted view on the highly conserved vertebrate PRNP and indicates that the SPRN gene may have more prominent function. More conserved Sprn could therefore substitute for the loss of less conserved, dispensable Prnp in the Prnp knock-out mice. Furthermore, the pathogenic potential of PRNP may be a consequence of relaxed evolutionary constraints.¶ Depth of comparative genomic analysis, strategy to understand biological function, depends on the number of species in comparison and their relative evolutionary distance. To understand better evolution and function of mammalian PRNP, I isolated and characterized the PRNP gene from Australian model marsupial tammar wallaby (Macropus eugenii). Marsupials are mammals separated from their eutherian relatives by roughly 180 million years. Comparison of the tammar wallaby and Brazilian opossum PrP with other vertebrate PrPs indicated patterns of evolution of the PrP regions. Whereas the repeat region is conserved within lineages but differs between lineages, the hydrophobic region is invariably conserved in all the PrPs. Conservation of PrP between marsupials and eutherians suggests that marsupial PrP could have the same pathogenic potential as eutherian PrPs. Using the marsupial PRNP gene in comparison with the PRNP genes from eutherian species in which prion diseases occur naturally (human, bovine, ovine) or experimentally (mouse), I defined gene regions that are conserved mammalian-wide and showed the utility of the marsupial genomic sequence for cross-species comparisons. These regions are potential regulatory elements that could govern gene expression and posttranscriptional control of mRNA activity. These findings shed new light on the normal function of mammalian PRNP supporting best the signal-transduction hypothesis. The normal function of PRNP may be triggering of signalling cascades which contribute to cell-cell interactions and may act anti-apoptotically. Yet, in the heterogenous set of cells expressing PrP^C these pathways will contribute to a number of cell-specific phenotypes, such as the synaptic plasticity and activation of lymphoid cells.
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Mead, Simon Harvey. "Molecular genetic analysis of the prion protein gene locus in human prion disease." Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.417947.
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Moore, Richard C. "Gene targeting studies at the mouse prion protein locus." Thesis, University of Edinburgh, 1997. http://hdl.handle.net/1842/11184.
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The prion protein (PrPc) is a normal host-encoded glycoprotein which accumulates as a disease specific protease-resistant isoform (PrPsc) in the brains of infected hosts. In a number of species PrP polymorphisms and germline mutations are associated with the modulation of disease phenotype and the occurrence of familial prion disease. To investigate the biological consequences of manipulation of the prion protein in mice a flexible two step double replacement gene targeting strategy was developed. This method can be used to generate a series of mouse lines with alterations to the mouse prion protein gene (Prn-p). To facilitate gene targeting studies a restriction map of the 129/Ola Prn-p locus was constructed and a series of overlapping genomic clones were retrieved from a λ DASH II bacteriophage 129/Ola library. The double replacement strategy was used to generate PrP deficient mice and mice with subtle alterations to PrP codons 108 and 189. Murine PrP 108F/V_189L/T dimorphisms give rise to 2 distinct PrP allotypes, PrP-A and PrP-B and these are postulated to be responsible for the control of incubation time following challenge with a wide range of prion inocula. To test this proposal the endogenous 129/Ola PrP-A allotype [108L_189T] was converted by gene targeting to encode the PrP-B allotype [108F_189V]. Mice bearing codon 108 and 189 alterations were challenged with mouse adapted BSE isolate 301V. Gene targeting in 129/Ola derived HM-1 ES cells and breeding with 129/Ola mice enabled the investigation of the effect of PrP alterations in the absence of PrP overexpression artefacts or the influence of non-Prn-p genes. The dramatic acceleration of incubation time in mice homozygous for the Prn-pa[108F_189V] gene targeted allele confirmed the major role of codons 108 and 189 in the control of BSE isolate 301V incubation time - and probably other prion isolates. This data provides the strongest evidence yet that the incubation time control, long attributed to the action of different alleles of Sinc (Prn-i), is determined by PrP codon 108L/F and 189T/V dimorphisms.
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Soldevila, Trepat Marta. "Genetic variation in humans and chimpanzees in the prion protein gene." Doctoral thesis, Universitat Pompeu Fabra, 2005. http://hdl.handle.net/10803/7189.
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En el gen de la proteïna priònica, o PRNP, hem observat que el particular patró de variació que hem trobat basant-nos en dades de seqüenciació en humans es deu a selecció positiva, i que el mètode utilitzat per detectar selecció és crític. Utilitzant dades basades en SNPs es pot introduir un biaix al aplicar tests de neutralitat basats en diversitat de seqüències, i això pot portar a conclusions errònies. A més, hem vist que els polimorfismes en els codons 129 i 219 presenten gran diferències de freqüència en diferents poblacions humanes i també hem vist que aquestes posicions estan fixades en ximpanzés. La variació trobada en controls ha estat comparada amb el patró de variació existent en pacients per la mateixa regió. La reseqüenciació del gen PRNP en un gran nombre de mostres humanes i de ximpanzés ens ha permès obtenir un gran nombre d´informació d´aquest gen.In the prion gene or PRNP, we have observed that the particular pattern of variation that we have found in this gene based on sequencing data in humans is due to positive selection, and that the method and the approach used to detect this selection critical. Ascertainment bias can be introduced by using SNP data and applying neutrality tests based on sequence diversity, therefore leading to anomalous conclusions being drawn. Moreover, we have seen that polymorphisms in codon 129 and 219 have big differences in frequency in different human populations and we have also seen that these positions are fixed in chimpanzees. The normal variation that we found in controls have been then compared with patients for the same region. The resequencing of PRNP in a very large sample of humans and chimpanzees has provided a great deal of information on this gene.
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Thumdee, Patama. "The prenatal expression of mRNA and protein of the prion protein gene, PRNP, in sheep." [S.l.] : [s.n.], 2007. http://deposit.ddb.de/cgi-bin/dokserv?idn=983755728.
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Uzun, Begum. "Surveillance Of Prion Protein (prp) Gene Polymorphisms In Turkish Native Sheep Breeds." Master's thesis, METU, 2012. http://etd.lib.metu.edu.tr/upload/12614353/index.pdf.
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v
It was found that most of the classical scrapie genotypes belong to R3 risk group,
whereas atypical scrapie genotypes belonging to zero (0) and one (1) risk groups were
frequently seen in sheep analyzed. In other words, Turkish sheep is found to have
intermediate risk of classical scrapie and low atypical scrapie risk, in general.
The data from the current study may help to establish a breeding program for classical
scrapie control in Turkey and will be beneficial for both the animal and public health in
the country. In addition, the outcomes of the study will fill the gap which is present in
the geographic distribution data of PrP gene polymorphisms in Eurasia.Scrapie is an infectious fatal disease of sheep and goats which affects the central nervous
system. In the present study, samples of 14 native Turkish sheep breeds (n=655) were
analyzed with respect to their polymorphisms of PrP gene (at codons: 136, 141, 154 and
171) and their classical and atypical scrapie risk levels were identified.
Turkish sheep are found to have the highest PrP genetic variability with 13 classical
scrapie alleles and 14 atypical scrapie alleles compared to all previous studies. Classical
scrapie-susceptible and wild-type ARQ allele was found as the most frequent allele in
Turkish sheep examined. The most classical scrapie-susceptible allele, VRQ was
detected at low frequencies in 5 of the breeds (Çine Ç
apari, Dagliç
, Kivircik, Karayaka and Gö
kç
eada). One novel allele (TL141HQ) was observed in Sakiz breed for the first time in this study.
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Mallucci, Giovanna Rachele. "Prion protein gene knockout in the mouse using the Cre/1oxP system." Thesis, Imperial College London, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.271231.
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Mahal, Sukhvir Paul. "Isolation and characterisation of the promoter region of the human prion protein gene." Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313746.
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Peralta, Oscar Alejandro. "Developmental Regulation of Prion Expression in Cattle and Mouse Embryonic Stem Cells." Diss., Virginia Tech, 2008. http://hdl.handle.net/10919/28584.
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The host encoded cellular prion protein (PrPC) is an N-linked glycoprotein tethered to the cell membrane by a glycophosphatidylinositol (GPI) anchor. Under certain conditions, PrPC can undergo conversion into a conformationally-altered isoform (PrPSc) widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Thus, tissues expressing PrPC are potential sites for conversion of PrPSc during TSE pathogenesis. Although much is known about the role of PrPSc in prion diseases, the normal function of PrPC is poorly understood. Lines of mice and cattle in which PrPC has been ablated by gene knockout show no major phenotypical alterations other than resistance to TSE infection. However, recent reports using Prnp-null mouse models have suggested the participation of PrPC in neural stem/progenitor cell proliferation and differentiation. The first objective in our study was to map the expression of PrPC in twenty six somatic and reproductive tissues in ruminants. Our second objective was to characterize the ontogeny of PrPC expression during bovine embryonic and early fetal development. Finally, we used a mouse embryonic stem cell (mESC) model to study the potential role of PrPC during neurogenesis. In adult tissues, intense expression of PrPC was detected in the central nervous system (CNS), thymus and testes, whereas the liver, striated muscle and female reproductive tissues showed the lowest expression. We observed that PrPC was associated with tissues undergoing cellular differentiation including spermatogenesis, lymphocyte activation and hair follicle regeneration. Analyses in bovine embryos and fetuses indicated peaks in expression of PrPC at days 4 and 18 post-fertilization, stages associated with the maternal-zygote transition and the maternal recognition of pregnancy and initiation of placental attachment, respectively. Later in development, PrPC was expressed in the CNS where it was localized in mature neurons of the neuroepithelium and emerging neural trunks. Based on these observations, we hypothesized that PrPC was involved in neurogenesis. We tested this hypothesis in a murine embryonic stem cell model (mESC). mESC were induced to form embryoid bodies (EBs) by placing them in suspension culture under differentiating conditions and allowed to differentiate in vitro for 20 days. We detected increasing levels of PrPC starting on day 12 (8.21- fold higher vs. day 0; P < 0.05) and continuing until day 20 (20.77-fold higher vs. day 0; P < 0.05). PrPC expression was negatively correlated with pluripotency marker Oct-4 (r= -0.85) confirming that mESC had indeed differentiated. To provide a more robust system for assessing the role of PrPC in neural differentiation, mESC were cultured with or without retinoic acid (RA) to encourage differentiation into neural lineages. Induction of EBs with retinoic acid (RA) resulted in an earlier up-regulation of PrPC and nestin (day 12 vs. day 16; P < 0.05). In addition, immunofluorescence studies indicated co-expression of PrPC and nestin in the same cells. The results of these experiments suggested a temporal link between PrPC expression and expression of nestin, a marker of neural progenitor cells. We next tested whether PrPC was involved in RA-enhanced neural differentiation from mESC using a PrPC knockdown model. Plasmid vectors designed to express either a PrP-targeted shRNA or scrambled, control shRNA were transfected into mESC. Stable transfectants were selected under G418 and cloned. PrP-targeted and control shRNA clones, as well as wild-type mESC, were differentiated in presence of RA and sampled as above. PrPC expression was knocked down in PrP-targeted shRNA cultures between days 12 and 20 (62.2 % average reduction vs. scrambled shRNA controls). Nestin expression was reduced at days 16 and 20 in PrPC knockdown cells (61.3% and 70.7%, respectively vs. scrambled shRNA controls). These results provide evidence that PrPC plays a role in the neural differentiation at a point up-stream from the stages at which nestin is expressed. In conclusion, the widely distributed expression of PrPC in ruminant tissues suggests an important biological role for this protein. In the present work we have provided evidence for the participation of PrPC in the differentiation of mESC along the neurogenic pathway.Ph. D.
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Ribeiro, Fernanda Trentini Lopes. "Polimorfismo do gene da proteína prion celular (prpc) e imunohistoquímica de tecido linfóide em ovinos = Polymorphism of cellular prion protein (PrPC) and immunohistochemistry of lymphoid tissue of sheep / Fernanda Trentini Lopes Ribeiro ; orientadora, Cristina Santos Sotomaior." reponame:Biblioteca Digital de Teses e Dissertações da PUC_PR, 2011. http://www.biblioteca.pucpr.br/tede/tde_busca/arquivo.php?codArquivo=2231.
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Dissertação (mestrado) - Pontifícia Universidade Católica do Paraná, São José dos Pinhais, 2011Inclui bibliografias
Scrapie é uma doença neurodegenerativa, progressiva e fatal de ovinos e caprinos, pertencente ao grupo das Encefalopatias Espongiformes Transmissíveis (EETs), ou doenças priônicas. O acúmulo de uma isoforma normal (PrPSc) da proteína prion celular (PrPC)
Scrapie is a fatal, neurodegenerative disease that affects sheep and goats and belongs to the Transmissible Spongiform Encephalopathies (TSEs) or prion diseases. It is caused by the deposition of an abnormal isoform (PrPSc) of the host-encoded cellular pr
Books on the topic "Prion protein gene"
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Loftus, Brendan. Analysis of the prion protein (PrP) and PrP genes from Ovis aries and Oryctalagus cuniculus. Dublin: University College Dublin, 1996.
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Mouillet-Richard, Sophie, and Jean-Luc Vilotte, eds. Promiscuous Functions of the Prion Protein Gene Family. Frontiers Media SA, 2015. http://dx.doi.org/10.3389/978-2-88919-605-0.
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Hope, James, and Mark P. Dagleish. Prion-protein-related diseases of animals and man. Oxford University Press, 2011. http://dx.doi.org/10.1093/med/9780198570028.003.0041.
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Scrapie, bovine spongiform encephalopathy (BSE), Creutzfeldt–Jakob disease (CJD), and related diseases of mink (transmissible mink encephalopathy), mule deer and elk (chronic wasting disease) are the founder members of a group of diseases called the transmissible degenerative (or spongiform) encephalopathies (TSE). These diseases can be transmitted by prions from affected to healthy animals by inoculation or by feeding diseased tissues. Prions are cellular proteins that can transfer metabolic and pathological phenotypes vertically from parent to progeny or horizontally between cells and animals. TSEs are characterised by the accumulation of the prion form of the mammalian prion protein (PrPC) in the central nervous system or peripheral tissues of animals and humans. Mutations of the human PrP gene are linked to rare, familial forms of disease and prion-protein gene polymorphisms in humans and other species are linked to survival time and disease characteristics in affected individuals. Iatrogenic transmission of CJD in man has occurred, and a variant form of CJD (vCJD) is due to cross-species transmission of BSE from cattle to humans. Atypical forms of scrapie and BSE have been identified during large-scale monitoring for TSEs worldwide. This chapter outlines our current understanding of scrapie, BSE, CJD and other TSEs and highlights recent progress in defining the role in disease of the prion protein, PrP.
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Mastrianni, James A., and Joshuae G. Gallardo. Prion Diseases. Oxford University Press, 2017. http://dx.doi.org/10.1093/med/9780199937837.003.0166.
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Prion diseases are transmissible fatal neurodegenerative disorders resulting from the accumulation of misfolded prion protein. Although primarily sporadic diseases, 5% to 10% result from a mutation of the prion protein gene (PRNP), and less than 1% occur from exposure to prions. The current family of prion diseases includes Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), fatal insomnia (FI), variant CJD (vCJD), and variably protease-sensitive prionopathy (VPSPr). Kuru is a disease of historical interest that was transmitted through cannibalistic rituals. Iatrogenic CJD (iCJD) is the result of secondary transmission of prion disease from contaminated biologicals.
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Gluckman, Sir Peter, Mark Hanson, Chong Yap Seng, and Anne Bardsley. Zinc in pregnancy and breastfeeding. Oxford University Press, 2015. http://dx.doi.org/10.1093/med/9780198722700.003.0025.
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Zinc is an essential mineral involved in gene expression, cell growth and division, neurotransmission, and reproductive and immune functions. It is crucial for periods of growth, including pregnancy and lactation, infancy, childhood, and adolescence. Daily requirements in pregnancy increase by ~40, so adequate zinc stores should be ensured prior to pregnancy so that the high requirements of the developing fetus can be met. Inadequate zinc intake often accompanies general protein/calorie malnourishment but is also seen in individuals consuming poor quality diets. Diets low in animal protein and/or high in phytates (e.g. in whole grains) provide limited amounts of bioavailable zinc. Prenatal vitamins containing zinc are advisable with such diets. Zinc is required for male fertility, and supplementation may improve fertility in subfertile men.
Book chapters on the topic "Prion protein gene"
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Nitrini, Ricardo, Sergio Rosemberg, Maria Rita Passos-Bueno, Luis S. Texeira da Silva, Paula Iughetti, Maria Papadopoulos, P. M. Carrilho, et al. "Human Prion Protein Gene Mutation at Codon 183 Associated with an Atypical Form of Prion Disease." In Prions and Brain Diseases in Animals and Humans, 25–32. Boston, MA: Springer US, 1998. http://dx.doi.org/10.1007/978-1-4899-1896-3_3.
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Gupta, N., S. Choudhary, G. Malik, A. Pandey, and S. C. Gupta. "Single Nucleotide Polymorphism (SNP) in Prion Protein Gene (PRNP) exon-3 in Gaddi Sheep." In Animal Genomics for Animal Health, 261–66. Basel: KARGER, 2008. http://dx.doi.org/10.1159/000317169.
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Zecevic, Ervin, Admir Dokso, Alma Rustempasic, and Muhamed Brka. "Polymorphisms of the ovine prion protein (PrP) Gene in the Pramenka Sheep Breed Population(s) in Bosnia and Herzegovina - Kupreski Strain." In 30th Scientific-Experts Conference of Agriculture and Food Industry, 109–16. Cham: Springer International Publishing, 2020. http://dx.doi.org/10.1007/978-3-030-40049-1_14.
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Oesch, B., D. Westaway, and S. B. Prusiner. "Prion Protein Genes: Evolutionary and Functional Aspects." In Current Topics in Microbiology and Immunology, 109–24. Berlin, Heidelberg: Springer Berlin Heidelberg, 1991. http://dx.doi.org/10.1007/978-3-642-76540-7_7.
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Lee, Inyoul, David Westaway, Arian Smit, Carol Cooper, Hong Yao, Stanley B. Prusiner, and Leroy Hood. "Large-Scale Sequencing of Human, Mouse, and Sheep Prion Protein Genes." In Prions and Brain Diseases in Animals and Humans, 59–75. Boston, MA: Springer US, 1998. http://dx.doi.org/10.1007/978-1-4899-1896-3_8.
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Kalejta, R. F. "Functions of Human Cytomegalovirus Tegument Proteins Prior to Immediate Early Gene Expression." In Current Topics in Microbiology and Immunology, 101–15. Berlin, Heidelberg: Springer Berlin Heidelberg, 2008. http://dx.doi.org/10.1007/978-3-540-77349-8_6.
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Pimenta, J., L. Lopes-da-Costa, C. C. Marques, J. P. Barbas, M. C. Baptista, and R. M. L. N. Pereira. "From Villains to Heroes: Insights into the Antagonizing Functions of Prion like Genes and Proteins." In Advances in Animal Health, Medicine and Production, 373–88. Cham: Springer International Publishing, 2020. http://dx.doi.org/10.1007/978-3-030-61981-7_20.
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Nihat, Akin, TzeHow Mok, and John Collinge. "Prion disease." In New Oxford Textbook of Psychiatry, edited by John R. Geddes, Nancy C. Andreasen, and Guy M. Goodwin, 414–23. Oxford University Press, 2020. http://dx.doi.org/10.1093/med/9780198713005.003.0042.
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Prion diseases are fatal neurodegenerative conditions that may arise sporadically or be inherited or acquired by environmental exposure to infectious prions—transmissible agents composed of multimeric assemblies of misfolded protein. The core clinical features are progressive cognitive decline, accompanied with ataxia, myoclonus, and pyramidal or extra-pramidal motor signs. While the most common form—sporadic Creutzfeldt–Jakob disease—is generally rapidly progressive over weeks or months, inherited prion diseases can span many years, with diverse clinical features readily mimicking other neurodegenerative diseases. Psychiatric features, including agitation, anxiety, depression, hallucinations, and behavioural disturbances, are common in the early stages. Diagnosis can usually be made with confidence by the combination of clinical criteria, diffusion-weighted magnetic resonance imaging, electroencephalogram, and specialized cerebrospinal fluid analysis. Inherited prion disease can be confirmed with prion protein gene analysis, which should be considered in all early-onset dementing and ataxic conditions. It is now becoming clear that the fundamental molecular pathogenesis—seeded protein polymerization—is relevant to other neurodegenerative diseases, notably Alzheimer’s disease.
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Ironside, James W. "Human Prion Diseases." In Escourolle and Poirier's Manual of Basic Neuropathology, 159–71. Oxford University Press, 2018. http://dx.doi.org/10.1093/med/9780190675011.003.0006.
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Human prion diseases include idiopathic, genetic, and acquired disorders. Heterogeneous clinicopathologic features make diagnosis challenging. Accurate diagnosis requires a combined clinical, neuropathologic, genetic, and biochemical approach. Neuropathologic assessment is performed following autopsy in most cases. The brain is sampled and studied by tinctorial stains and immunohistochemistry for disease-associated form of the prion protein. Unfixed frozen brain tissue is retained for Western blot analysis of protease-resistant prion protein isoform and for DNA extraction to sequence the prion protein gene. Assessment of spongiform change, gliosis neuronal loss, and accumulation of disease-associated prion protein in the brain can help to determine major categories of human prion disease. Additional clinical, genetic, and biochemical data allow diagnosis and subclassification into disease subtypes, particularly in sporadic Creutzfeldt–Jakob disease. Neuropathology continues to play a role in the recognition and understanding of the expanding spectrum of human prion disease and identification of disease variants that may emerge in the future.
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Urwin, Patrick JM, and Anna M. Molesworth. "The neuroepidemiology of human prion disease." In Oxford Textbook of Neurologic and Neuropsychiatric Epidemiology, edited by Carol Brayne, Valery L. Feigin, Lenore J. Launer, and Giancarlo Logroscino, 367–78. Oxford University Press, 2020. http://dx.doi.org/10.1093/med/9780198749493.003.0035.
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Human prion diseases comprise a number of rare and fatal neurodegenerative conditions that result from the accumulation in the central nervous system of an abnormal form of a naturally occurring protein, called the prion protein. The diseases occur in genetic, sporadic, and acquired forms: genetic disease is associated with mutations in the prion protein gene (PRNP); sporadic disease is thought to result from a spontaneous protein misfolding event; acquired disease results from transmission of infection from an animal or another human. The potential transmissibility of the prion in any of these forms, either in disease states or during the incubation period, has implications for public health. Here we focus on Creutzfeldt-Jakob Disease (CJD), including variant Creutzfeldt-Jakob Disease (vCJD), although we will also discuss other forms of human prion disease.
Conference papers on the topic "Prion protein gene"
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GHETTI, BERNARDINO, LETICIA MIRAVALLE, KEIJI YAMAGUCHI, FRANCINE EPPERSON, JILL R. MURRELL, TONY PERKINS, SIU HUI, et al. "ROLE OF THE POLYMORPHISM AT CODON 129 OF THE PRION PROTEIN GENE IN THE PHENOTYPIC EXPRESSION OF GERSTMANN-STRÄUSSLER-SCHEINKER DISEASE ASSOCIATED WITH THE F198S MUTATION." In The 32nd Session of International Seminars and International Collaboration. WORLD SCIENTIFIC, 2005. http://dx.doi.org/10.1142/9789812701787_0015.
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Stoicescu, Ramona, Razvan-Alexandru Stoicescu, Codrin Gheorghe, Adina Honcea, and Iulian Bratu. "CONSIDERATIONS ON SARS-COV-2 DIAGNOSIS IN THE LABORATORY OF UNIVERSITY EMERGENCY CLINICAL HOSPITAL OF CONSTANTA." In GEOLINKS Conference Proceedings. Saima Consult Ltd, 2021. http://dx.doi.org/10.32008/geolinks2021/b1/v3/07.
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Coronaviruses are members of the Coronaviridae family. They are enveloped, non-segmented, positive-sense, single-stranded RNA viruses. Their genome size is about 30 kilobases (kb) which consist, at the 5’ end, of non-structural open reading frames (ORFs: ORF1a, ORF 1b) which code for 16 non structural proteins, and at the 3’ end the genes which code for four structural proteins including membrane (M), envelope (E), spike (S), and nucleocapsid (N) proteins. Due to the rapid spread of COVID-19, a reliable detection method is needed for patient diagnosis especially in the early stages of the disease. WHO has recommended nucleic acid amplification tests such as real-time reverse transcription-polymerase chain reaction (RT-PCR). The assay detects three SARS-CoV-2 RNA targets: the envelope (E) gene, the nucleocapsid (N) gene and a region of the open reading frame (ORF1) of the RNA-dependent RNA polymerase (RdRp) gene from SARS-CoV-2 virus isolate Wuhan-Hu-1. Our study was made in the first 3 months of the year 2021 using the real-time RT PCR results obtained in the Cellular Biology ward of the University Emergency Clinical Hospital. In our lab we are testing the inpatients from the hospital wards (Neurology, Pediatrics, Surgery, Internal medicine, ICU, Cardiology, etc.); we are also testing the outpatients from Dialysis and Oncology, 2 days prior to their therapy; we also test the health care personnel. The number of tests we performed was: in January 1456, with 399 positive results (27.4%), 33 deaths; in February 1273 tests, 221 positive (17.36%), 16 deaths; in March 1471 tests, 373 positive (25.36%), 37 deceased.
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Jiang, Chunheng, Jianxi Gao, and Malik Magdon-Ismail. "Inferring Degrees from Incomplete Networks and Nonlinear Dynamics." In Twenty-Ninth International Joint Conference on Artificial Intelligence and Seventeenth Pacific Rim International Conference on Artificial Intelligence {IJCAI-PRICAI-20}. California: International Joint Conferences on Artificial Intelligence Organization, 2020. http://dx.doi.org/10.24963/ijcai.2020/457.
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Inferring topological characteristics of complex networks from observed data is critical to understand the dynamical behavior of networked systems, ranging from the Internet and the World Wide Web to biological networks and social networks. Prior studies usually focus on the structure-based estimation to infer network sizes, degree distributions, average degrees, and more. Little effort attempted to estimate the specific degree of each vertex from a sampled induced graph, which prevents us from measuring the lethality of nodes in protein networks and influencers in social networks. The current approaches dramatically fail for a tiny sampled induced graph and require a specific sampling method and a large sample size. These approaches neglect information of the vertex state, representing the dynamical behavior of the networked system, such as the biomass of species or expression of a gene, which is useful for degree estimation. We fill this gap by developing a framework to infer individual vertex degrees using both information of the sampled topology and vertex state. We combine the mean-field theory with combinatorial optimization to learn vertex degrees. Experimental results on real networks with a variety of dynamics demonstrate that our framework can produce reliable degree estimates and dramatically improve existing link prediction methods by replacing the sampled degrees with our estimated degrees.
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Li, Yongqiang, Jennifer Quincy, Scott W. Case, David A. Dillard, Michael Budinski, and Yeh-Hung Lai. "Using a Knife Slitting Test to Characterize the Fracture Resistance of Proton Exchange Membranes." In ASME 2006 4th International Conference on Fuel Cell Science, Engineering and Technology. ASMEDC, 2006. http://dx.doi.org/10.1115/fuelcell2006-97096.
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Through-the-thickness flaws or “pinholes” in proton exchange membranes (PEM) can lead to gas crossover, reducing fuel cell efficiency, accelerating degradation, and raising safety issues. The multi-physics process that causes these flaws is not fully understood, but stress state, environmental exposure, and cyclic operation may all be contributing factors. Fracture mechanics has proven to be useful in characterizing degradation of many materials, including polymers subjected to environmental challenges. Although unclear if pinhole formation can be successfully characterized and predicted from a fracture perspective, this study continues our prior work to characterize PEMs in such a manner. Because of the lack of constraint, thin films often exhibit very high fracture energies and large plastic zones, features that are not consistent with observations of PEM failures. In an effort to obtain the fracture energy with very little dissipation, knife-slitting tests were conducted to reduce the crack tip plasticity. With modifications made to the systems used by Wang and Gent (1994) and by Dillard et al (2005), a slitter that maintains a constant tearing angle during the slitting process was developed. While fracture energies on the order of 104J/m2 were measured with double edge notched test samples, and on the order of 103J/m2 were measured with trouser tear samples, the knife slit test resulted in fracture energies as low as several hundred J/m2. An environmental chamber was used to enclose the slitting process so experiments at elevated temperature and moisture levels could be conducted. The relevance of these fracture energies to observed PEM failures in operating fuel cells is not fully understood. Nonetheless, the ability to obtain fracture energies approaching the intrinsic fracture energy of these ductile membranes is believed to be useful in studying what appear to be more brittle fracture modes that have been observed in PEMs.
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A. Buzzetto-Hollywood, Nicole, Austin J. Hill, and Troy Banks. "Early Findings of a Study Exploring the Social Media, Political and Cultural Awareness, and Civic Activism of Gen Z Students in the Mid-Atlantic United States [Abstract]." In InSITE 2021: Informing Science + IT Education Conferences. Informing Science Institute, 2021. http://dx.doi.org/10.28945/4762.
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Aim/Purpose: This paper provides the results of the preliminary analysis of the findings of an ongoing study that seeks to examine the social media use, cultural and political awareness, civic engagement, issue prioritization, and social activism of Gen Z students enrolled at four different institutional types located in the Mid-Atlantic region of the United States. The aim of this study is to look at the group as a whole as well as compare findings across populations. The institutional types under consideration include a mid-sized majority serving or otherwise referred to as a traditionally white institution (TWI) located in a small coastal city on the Atlantic Ocean, a small Historically Black University (HBCU) located in a rural area, a large community college located in a county that is a mixture of rural and suburban and which sits on the border of Maryland and Pennsylvania, and graduating high school students enrolled in career and technical education (CTE) programs in a large urban area. This exploration is purposed to examine the behaviors and expectations of Gen Z students within a representative American region during a time of tremendous turmoil and civil unrest in the United States. Background: Over 74 million strong, Gen Z makes up almost one-quarter of the U.S. population. They already outnumber any current living generation and are the first true digital natives. Born after 1996 and through 2012, they are known for their short attention spans and heightened ability to multi-task. Raised in the age of the smart phone, they have been tethered to digital devices from a young age with most having the preponderance of their childhood milestones commemorated online. Often called Zoomers, they are more racially and ethnically diverse than any previous generation and are on track to be the most well-educated generation in history. Gen Zers in the United States have been found in the research to be progressive and pro-government and viewing increasing racial and ethnic diversity as positive change. Finally, they are less likely to hold xenophobic beliefs such as the notion of American exceptionalism and superiority that have been popular with by prior generations. The United States has been in a period of social and civil unrest in recent years with concerns over systematic racism, rampant inequalities, political polarization, xenophobia, police violence, sexual assault and harassment, and the growing epidemic of gun violence. Anxieties stirred by the COVID-19 pandemic further compounded these issues resulting in a powder keg explosion occurring throughout the summer of 2020 and leading well into 2021. As a result, the United States has deteriorated significantly in the Civil Unrest Index falling from 91st to 34th. The vitriol, polarization, protests, murders, and shootings have all occurred during Gen Z’s formative years, and the limited research available indicates that it has shaped their values and political views. Methodology: The Mid-Atlantic region is a portion of the United States that exists as the overlap between the northeastern and southeastern portions of the country. It includes the nation’s capital, as well as large urban centers, small cities, suburbs, and rural enclaves. It is one of the most socially, economically, racially, and culturally diverse parts of the United States and is often referred to as the “typically American region.” An electronic survey was administered to students from 2019 through 2021 attending a high school dual enrollment program, a minority serving institution, a majority serving institution, and a community college all located within the larger mid-Atlantic region. The survey included a combination of multiple response, Likert scaled, dichotomous, open ended, and ordinal questions. It was developed in the Survey Monkey system and reviewed by several content and methodological experts in order to examine bias, vagueness, or potential semantic problems. Finally, the survey was pilot tested prior to implementation in order to explore the efficacy of the research methodology. It was then modified accordingly prior to widespread distribution to potential participants. The surveys were administered to students enrolled in classes taught by the authors all of whom are educators. Participation was voluntary, optional, and anonymous. Over 800 individuals completed the survey with just over 700 usable results, after partial completes and the responses of individuals outside of the 18-24 age range were removed. Findings: Participants in this study overwhelmingly were users of social media. In descending order, YouTube, Instagram, Snapchat, Twitter, Facebook, Pinterest, WhatsApp, LinkedIn and Tik Tok were the most popular social media services reported as being used. When volume of use was considered, Instagram, Snapchat, YouTube and Twitter were the most cited with most participants reporting using Instagram and Snapchat multiple times a day. When asked to select which social media service they would use if forced to choose just one, the number one choice was YouTube followed by Instagram and Snapchat. Additionally, more than half of participants responded that they have uploaded a video to a video sharing site such as YouTube or Tik Tok. When asked about their familiarity with different technologies, participants overwhelmingly responded that they are “very familiar” with smart phones, searching the Web, social media, and email. About half the respondents said that they were “very familiar” with common computer applications such as the Microsoft Office Suite or Google Suite with another third saying that they were “somewhat familiar.” When asked about Learning Management Systems (LMS) like Blackboard, Course Compass, Canvas, Edmodo, Moodle, Course Sites, Google Classroom, Mindtap, Schoology, Absorb, D2L, itslearning, Otus, PowerSchool, or WizIQ, only 43% said they were “very familiar” with 31% responding that they were “somewhat familiar.” Finally, about half the students were either “very” or “somewhat” familiar with operating systems such as Windows. A few preferences with respect to technology in the teaching and learning process were explored in the survey. Most students (85%) responded that they want course announcements and reminders sent to their phones, 76% expect their courses to incorporate the use of technology, 71% want their courses to have course websites, and 71% said that they would rather watch a video than read a book chapter. When asked to consider the future, over 81% or respondents reported that technology will play a major role in their future career. Most participants considered themselves “informed” or “well informed” about current events although few considered themselves “very informed” or “well informed” about politics. When asked how they get their news, the most common forum reported for getting news and information about current events and politics was social media with 81% of respondents reporting. Gen Z is known to be an engaged generation and the participants in this study were not an exception. As such, it came as no surprise to discover that, in the past year more than 78% of respondents had educated friends or family about an important social or political issue, about half (48%) had donated to a cause of importance to them, more than a quarter (26%) had participated in a march or rally, and a quarter (26%) had actively boycotted a product or company. Further, about 37% consider themselves to be a social activist with another 41% responding that aren’t sure if they would consider themselves an activist and only 22% saying that they would not consider themselves an activist. When asked what issues were important to them, the most frequently cited were Black Lives Matter (75%), human trafficking (68%), sexual assault/harassment/Me Too (66.49%), gun violence (65.82%), women’s rights (65.15%), climate change (55.4%), immigration reform/deferred action for childhood arrivals (DACA) (48.8%), and LGBTQ+ rights (47.39%). When the schools were compared, there were only minor differences in social media use with the high school students indicating slightly more use of Tik Tok than the other participants. All groups were virtually equal when it came to how informed they perceived themselves about current events and politics. Consensus among groups existed with respect to how they get their news, and the community college and high school students were slightly more likely to have participated in a march, protest, or rally in the last 12 months than the university students. The community college and high school students were also slightly more likely to consider themselves social activists than the participants from either of the universities. When the importance of the issues was considered, significant differences based on institutional type were noted. Black Lives Matter (BLM) was identified as important by the largest portion of students attending the HBCU followed by the community college students and high school students. Less than half of the students attending the TWI considered BLM an important issue. Human trafficking was cited as important by a higher percentage of students attending the HBCU and urban high school than at the suburban and rural community college or the TWI. Sexual assault was considered important by the majority of students at all the schools with the percentage a bit smaller from the majority serving institution. About two thirds of the students at the high school, community college, and HBCU considered gun violence important versus about half the students at the majority serving institution. Women’s rights were reported as being important by more of the high school and HBCU participants than the community college or TWI. Climate change was considered important by about half the students at all schools with a slightly smaller portion reporting out the HBCU. Immigration reform/DACA was reported as important by half the high school, community college, and HBCU participants with only a third of the students from the majority serving institution citing it as an important issue. With respect to LGBTQ rights approximately half of the high school and community college participants cited it as important, 44.53% of the HBCU students, and only about a quarter of the students attending the majority serving institution. Contribution and Conclusion: This paper provides a timely investigation into the mindset of generation Z students living in the United States during a period of heightened civic unrest. This insight is useful to educators who should be informed about the generation of students that is currently populating higher education. The findings of this study are consistent with public opinion polls by Pew Research Center. According to the findings, the Gen Z students participating in this study are heavy users of multiple social media, expect technology to be integrated into teaching and learning, anticipate a future career where technology will play an important role, informed about current and political events, use social media as their main source for getting news and information, and fairly engaged in social activism. When institutional type was compared the students from the university with the more affluent and less diverse population were less likely to find social justice issues important than the other groups. Recommendations for Practitioners: During disruptive and contentious times, it is negligent to think that the abounding issues plaguing society are not important to our students. Gauging the issues of importance and levels of civic engagement provides us crucial information towards understanding the attitudes of students. Further, knowing how our students gain information, their social media usage, as well as how informed they are about current events and political issues can be used to more effectively communicate and educate. Recommendations for Researchers: As social media continues to proliferate daily life and become a vital means of news and information gathering, additional studies such as the one presented here are needed. Additionally, in other countries facing similarly turbulent times, measuring student interest, awareness, and engagement is highly informative. Impact on Society: During a highly contentious period replete with a large volume of civil unrest and compounded by a global pandemic, understanding the behaviors and attitudes of students can help us as higher education faculty be more attuned when it comes to the design and delivery of curriculum. Future Research This presentation presents preliminary findings. Data is still being collected and much more extensive statistical analyses will be performed.
Reports on the topic "Prion protein gene"
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Dubcovsky, Jorge, Tzion Fahima, Ann Blechl, and Phillip San Miguel. Validation of a candidate gene for increased grain protein content in wheat. United States Department of Agriculture, January 2007. http://dx.doi.org/10.32747/2007.7695857.bard.
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High Grain Protein Content (GPC) of wheat is important for improved nutritional value and industrial quality. However, selection for this trait is limited by our poor understanding of the genes involved in the accumulation of protein in the grain. A gene with a large effect on GPC was detected on the short arm of chromosome 6B in a Triticum turgidum ssp. dicoccoides accession from Israel (DIC, hereafter). During the previous BARD project we constructed a half-million clones Bacterial Artificial Chromosome (BAC) library of tetraploid wheat including the high GPC allele from DIC and mapped the GPC-B1 locus within a 0.3-cM interval. Our long-term goal is to provide a better understanding of the genes controlling grain protein content in wheat. The specific objectives of the current project were to: (1) complete the positional cloning of the GPC-B1 candidate gene; (2) characterize the allelic variation and (3) expression profile of the candidate gene; and (4) validate this gene by using a transgenic RNAi approach to reduce the GPC transcript levels. To achieve these goals we constructed a 245-kb physical map of the GPC-B1 region. Tetraploid and hexaploid wheat lines carrying this 245-kb DIC segment showed delayed senescence and increased GPC and grain micronutrients. The complete sequencing of this region revealed five genes. A high-resolution genetic map, based on approximately 9,000 gametes and new molecular markers enabled us to delimit the GPC-B1 locus to a 7.4-kb region. Complete linkage of the 7.4-kb region with earlier senescence and increase in GPC, Zn, and Fe concentrations in the grain suggested that GPC-B1 is a single gene with multiple pleiotropic effects. The annotation of this 7.4-kb region identified a single gene, encoding a NAC transcription factor, designated as NAM-B1. Allelic variation studies demonstrated that the ancestral wild wheat allele encodes a functional NAC transcription factor whereas modern wheat varieties carry a non-functional NAM-B1 allele. Quantitative PCR showed that transcript levels for the multiple NAMhomologues were low in flag leaves prior to anthesis, after which their levels increased significantly towards grain maturity. Reduction in RNA levels of the multiple NAMhomologues by RNA interference delayed senescence by over three weeks and reduced wheat grain protein, Zn, and Fe content by over 30%. In the transgenic RNAi plants, residual N, Zn and Fe in the dry leaves was significantly higher than in the control plants, confirming a more efficient nutrient remobilization in the presence of higher levels of GPC. The multiple pleiotropic effects of NAM genes suggest a central role for these genes as transcriptional regulators of multiple processes during leaf senescence, including nutrient remobilization to the developing grain. The cloning of GPC-B1 provides a direct link between the regulation of senescence and nutrient remobilization and an entry point to characterize the genes regulating these two processes. This may contribute to their more efficient manipulation in crops and translate into food with enhanced nutritional value. The characterization of the GPC-B1 gene will have a significant impact on wheat production in many regions of the world and will open the door for the identification of additional genes involved in the accumulation of protein in the grain.
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Tucker, Mark L., Shimon Meir, Amnon Lers, Sonia Philosoph-Hadas, and Cai-Zhong Jiang. Elucidation of signaling pathways that regulate ethylene-induced leaf and flower abscission of agriculturally important plants. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7597929.bard.
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The Problem: Abscission is a highly regulated process, occurring as a natural terminal stage of development, in which various organs are separated from the parent plant. In most plant species, the process is initiated by a decrease in active auxin in the abscission zone (AZ) and an increase in ethylene, and may be accelerated by postharvest or environmental stresses. Another potential key regulator in abscission is IDA (Inflorescence Deficient in Abscission), which was identified as an essential peptide signal for floral organ abscission in Arabidopsis. However, information is still lacking regarding the molecular mechanisms integrating all these regulators. In our previous BARD funded research we made substantial progress towards understanding these molecular events in tomato, and the study is still in progress. We established a powerful platform for analysis of genes for regulatory proteins expressed in AZ. We identified changes in gene expression for several transcription factors (TFs) directly linked to ethylene and auxin signaling and several additional regulatory proteins not so obviously linked to these hormones. Moreover, we demonstrated using a virus-induced gene silencing (VIGS) assay that several play a functional role in the onset of abscission. Based on these results we have selected 14 genes for further analysis in stably transformed tomato plants. All 14 genes were suppressed by RNA interference (RNAi) using a constitutive promoter, and 5 of them were also suppressed using an abscission-specific promoter. Transformations are currently at different stages of progress including some lines that already display an abscission phenotype. Objectives: We propose here to (1) complete the functional analysis of the stably transformed tomato plants with T2 lines and perform transcriptome analysis using custom abscission-specific microarrays; (2) conduct an indepth analysis of the role of IDA signaling in tomato leaf and flower abscission; (3) perform transcriptome and proteome analyses to extend the earlier gene expression studies to identify transcripts and proteins that are highly specific to the separation layer (i.e., target cells for cell separation) prior to the onset of abscission; (4) extend and compliment the work in tomato using a winnowed set of genes in soybean. Methodology: Next Generation Sequencing (NGS) of mRNA will be used to further increase the list of abscission-associated genes, and for preparation of a custom tomato abscission microarray to test altered gene expression in transgenic plants. Tandem mass spectrometry (LC-MS/MS) of protein extracts from leaf petiole, flower pedicel and their AZ tissues will be used to identify the proteome of the AZ before and during abscission. AZ-specific gene promoters will be used in stably transformed tomato plants to reduce non-target phenotypes. The bean pod mottle virus (BPMV) plasmid vectors will be used for VIGS analysis in soybean. Expected Contribution: Our study will provide new insights into the regulation of ethylene-induced abscission by further revealing the role of key regulators in the process. This will permit development of novel techniques for manipulating leaf and flower abscission, thereby improving the postharvest performance of agriculturally important crops.
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Barash, Itamar, J. Mina Bissell, Alexander Faerman, and Moshe Shani. Modification of Milk Composition via Transgenesis: The Role of the Extracellular Matrix in Regulating Transgene Expression. United States Department of Agriculture, July 1995. http://dx.doi.org/10.32747/1995.7570558.bard.
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Altering milk composition via transgenesis depends on three main factors. (1) The availability of an efficient regulatory sequences for targeting transgene(s) to the mammary gland; (2) a reliable in vitro model to test the expression of transgenes prior to their introduction to the animal genome; and (3) better understanding of the major factors which determine the rate of gene expression and protein synthesis. The current studies provide the necessary means and knowledge to alter milk protein composition via transgenesis. The following specific goals were achieved: a: Identifying regulatory regions in the b-lactoglobulin (BLG) gene and the cross-talk between elements which enabled us to construct an efficient vector for the expression of desirable cDNA's in the mammary gland. b: The establishment of a sheep mammary cell line that serves as a model for the analysis of endogenous and exogenous milk protein synthesis in the mammary gland of livestock. c: An accurate comparison of the potency of the 5' regulatory sequences from the BLG and whey acidic protein (WAP) promoters in directing the expression of human serum albumin (HSA) to the mammary gland in vitro and in vivo. In this study we have also shown that sequences within the coding region may determine a specific pattern of expression for the transgene, distinct from that of the native milk protein genes. d: Characterizing the dominant role of ECM in transgene expression in mammary epithelial cells. e: Further characterization of the BCE-1 enhancer element in the promoter of the b-casein gene as a binding site for the c/EBP-b and Stat5. Identifying its interaction with chromatin and its up regulation by inhibitors of histone deacetylation. f: Identifying a mechanism of translational control as a mediator for the synergistic effect of insulin and prolactin on protein synthesis in the mammary gland.
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Grafi, Gideon, and Brian Larkins. Endoreduplication in Maize Endosperm: An Approach for Increasing Crop Productivity. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7575285.bard.
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The focus of this research project is to investigate the role of endoreduplication in maize endosperm development and the extent to which this process contributes to high levels of starch and storage protein synthesis. Although endoreduplication has been widely observed in many cells and tissues, especially those with high levels of metabolic activity, the molecular mechanisms through which the cell cycle is altered to produce consecutive cycles of S-phase without an intervening M-phase are unknown. Our previous research has shown that changes in the expression of several cell cycle regulatory genes coincide with the onset of endoreduplication. During this process, there is a sharp reduction in the activity of the mitotic cyclin-dependent kinase (CDK) and activation of the S-phase CDK. It appears the M-phase CDK is stable, but its activity is blocked by a proteinaceous inhibitor. Coincidentally, the S-phase checkpoint protein, retinoblastoma (ZmRb), becomes phosphorylated, presumably releasing an E2F-type transcriptional regulator which promotes the expression of genes responsible for DNA synthesis. To investigate the role of these cell cycle proteins in endoreduplication, we have created transgenic maize plants that express various genes in an endosperm-specific manner using a storage protein (g-zein) promoter. During the first year of the grant, we constructed point mutations of the maize M-phase kinase, p34cdc2. One alteration replaced aspartic acid at position 146 with asparagine (p3630-CdcD146N), while another changed threonine 161 to alanine (p3630-CdcT161A). These mutations abolish the activity of the CDK. We hypothesized that expression of the mutant forms of p34cdc2 in endoreduplicating endosperm, compared to a control p34cdc2, would lead to extra cycles of DNA synthesis. We also fused the gene encoding the regulatory subunit of the M- phase kinase, cyclin B, under the g-zein promoter. Normally, cyclin B is expected to be destroyed prior to the onset of endoreduplication. By producing high levels of this protein in developing endosperm, we hypothesized that the M-phase would be extended, potentially reducing the number of cycles of endoreduplication. Finally, we genetically engineered the wheat dwarf virus RepA protein for endosperm-specific expression. RepA binds to the maize retinoblastoma protein and presumably releases E2F-like transcription factors that activate DNA synthesis. We anticipated that inactivation of ZmRb by RepA would lead to additional cycles of DNA synthesis.
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Heifetz, Yael, and Michael Bender. Success and failure in insect fertilization and reproduction - the role of the female accessory glands. United States Department of Agriculture, December 2006. http://dx.doi.org/10.32747/2006.7695586.bard.
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The research problem. Understanding of insect reproduction has been critical to the design of insect pest control strategies including disruptions of mate-finding, courtship and sperm transfer by male insects. It is well known that males transfer proteins to females during mating that profoundly affect female reproductive physiology, but little is known about the molecular basis of female mating response and no attempts have yet been made to interfere with female post-mating responses that directly bear on the efficacy of fertilization. The female reproductive tract provides a crucial environment for the events of fertilization yet thus far those events and the role of the female tract in influencing them are poorly understood. For this project, we have chosen to focus on the lower reproductive tract because it is the site of two processes critical to reproduction: sperm management (storage, maintenance, and release from storage) and fertilization. E,fforts during this project period centered on the elucidation of mating responses in the female lower reproductive tract The central goals of this project were: 1. To identify mating-responsive genes in the female lower reproductive tract using DNA microarray technology. 2. In parallel, to identify mating-responsive genes in these tissues using proteomic assays (2D gels and LC-MS/MS techniques). 3. To integrate proteomic and genomic analyses of reproductive tract gene expression to identify significant genes for functional analysis. Our main achievements were: 1. Identification of mating-responsive genes in the female lower reproductive tract. We identified 539 mating-responsive genes using genomic and proteomic approaches. This analysis revealed a shift from gene silencing to gene activation soon after mating and a peak in differential gene expression at 6 hours post-mating. In addition, comparison of the two datasets revealed an expression pattern consistent with the model that important reproductive proteins are pre-programmed for synthesis prior to mating. This work was published in Mack et al. (2006). Validation experiments using real-time PCR techniques suggest that microarray assays provide a conservativestimate of the true transcriptional activity in reproductive tissues. 2.lntegration of proteomics and genomics data sets. We compared the expression profiles from DNA microarray data with the proteins identified in our proteomic experiments. Although comparing the two data sets poses analyical challenges, it provides a more complete view of gene expression as well as insights into how specific genes may be regulated. This work was published in Mack et al. (2006). 3. Development of primary reproductive tract cell cultures. We developed primary cell cultures of dispersed reproductive tract cell types and determined conditions for organ culture of the entire reproductive tract. This work will allow us to rapidly screen mating-responsive genes for a variety of reproductive-tract specifi c functions. Scientific and agricultural significance. Together, these studies have defined the genetic response to mating in a part of the female reproductive tract that is critical for successful fertllization and have identified alarge set of mating-responsive genes. This work is the first to combine both genomic and proteomic approaches in determining female mating response in these tissues and has provided important insights into insect reproductive behavior.
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Dickman, Martin B., and Oded Yarden. Role of Phosphorylation in Fungal Spore Germination. United States Department of Agriculture, August 1993. http://dx.doi.org/10.32747/1993.7568761.bard.
Full textAbstract:
Spore germination is a common and fundamental event in fungal development and in many instances an essential phase of fungal infection and dissemination. Spore germination is also critical for hyperparasites to function as biocontrol agents as well as in fermentation proceses. Our common objective is to understand the mechanisms which regulated spore germination and identify factors involved in pathogenicity related prepenetration development. Our approach is to exploit the overall similarity among filamentous fungi using both a plant pathogen (Colletotricum trifolii) and a model system that is genetically sophisticated (Neurospora crassa). The simulataneous use of two organisms has the advantage of the available tools in Neurospora to rapidly advance the functional analysis of genes involved in spore germination and development of an economically important fungal phytopathogen. Towards this we have isolated a protein kinase gene from C. trifolii (TB3) that is maximally expressed during the first hour of conidial germination and prior to any visible gene tube formation. Based on sequence similarities with other organisms, this gene is likely to be involved in the proliferative response in the fungus. In addition, TB3 was able to functionally complement a N. crassa mutant (COT-1). Pharmacological studies indicated the importance of calmodulin in both germination and appressorium differentiation. Using an antisense vector from N. crassa, direct inhibition of calmodulin results in prevention of differentiation as well as pathogenicity. Both cAMP dependent protein kinase (PKA) and protein kinase C (PKC) like genes have been cloned from C. trifolii. Biochemical inhibition of PKA prevents germination; biochemical inhibitors of PKC prevents appressorium differentiation. In order to analyze reversible phosphorylation as a regulatory mechanism, some ser.thr dephosphorylative events have also been analyzed. Type 2A and Type 2B (calcineurin) phosphatases have been identified and structurally and functionally analyzed in N. crassa during this project. Both phosphatases are essential for hyphal growth and maintenance of proper hyphal architecture. In addition, a first novel-type (PPT/PP5-like) ser/thr phosphatase has been identified in a filamentous fungus. The highly collaborative project has improved our understanding of a fundamental process in fungi, and has identified targets which can be used to develop new approaches for control of fungal plant pathogens as well as improve the performance of beneficial fungi in the field and in industry. In addition, the feasibility of molecular technology transfer in comparative mycology has been demonstrated.
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Or, Etti, Tai-Ping Sun, Amnon Lichter, and Avichai Perl. Characterization and Manipulation of the Primary Components in Gibberellin Signaling in the Grape Berry. United States Department of Agriculture, January 2010. http://dx.doi.org/10.32747/2010.7592649.bard.
Full textAbstract:
Seedless cultivars dominate the table grape industry. In these cultivars it is mandatory to apply gibberellin (GA) to stimulate berry development to a commercially acceptable size. These cultivars differ in their sensitivity to GA application, and it frequently results in adverse effects such as decreased bud fertility and increased fruit drop. Our long term goals are to (1) understand the molecular basis for the differential sensitivity and identify markers for selection of sensitive cultivars (2) to develop new strategies for targeted manipulation of the grape berry response to GA that will eliminate the need in GA application and the undesirable effects of GA on the vine, while maintaining its desirable effects on the berry. Both strategies are expected to reduce production cost and meet growing consumer demand for reduced use of chemicals. This approach relies on a comprehensive characterization of the central components in the GA signaling cascade in the berry. Several key components in the GA signaling pathway were identified in Arabidopsis and rice, including the GA receptors, GID1s, and a family of DELLA proteins that are the major negative regulators of the GA response. GA activates its response pathway by binding to GID1s, which then target DELLAs for degradation via interaction with SLY, a DELLA specific F-box protein. In grape, only one DELLA gene was characterized prior to this study, which plays a major role in inhibiting GA-promoted stem growth and GA-repressed floral induction but it does not regulate fruit growth. Therefore, we speculated that other DELLA family member(s) may control GA responses in berry, and their identification and manipulation may result in GA-independent berry growth. In the current study we isolated two additional VvDELLA family members, two VvGID1 genes and two VvSLY genes. Arabidopsis anti-AtRGA polyclonal antibodies recognized all three purified VvDELLA proteins, but its interaction with VvDELLA3 was weaker. Overexpression of the VvDELLAs, the VvGID1s, and the VvSLYs in the Arabidopsis mutants ga1-3/rga-24, gid1a-2/1c-2 and sly1-10, respectively, rescued the various mutant phenotypes. In vitro GAdependent physical interaction was shown between the VvDELLAs and the VvGID1s, and GAindependent interaction was shown between the VvDELLAs and VvSLYs. Interestingly, VvDELLA3 did not interact with VvGID1b. Together, the results indicate that the identified grape homologs serve as functional DELLA repressors, receptors and DELLA-interacting F-box proteins. Expression analyses revealed that (1) VvDELLA2 was expressed in all the analyzed tissues and was the most abundant (2) VvDELLA1 was low expressed in berries, confirming former study (3) Except in carpels and very young berries, VvDELLA3 levels were the lowest in most tissues. (4) Expression of both VvGID1s was detected in all the grape tissues, but VvGID1b transcript levels were significantly higher than VvGID1a. (5) In general, both VvDELLAs and VvGID1s transcripts levels increased as tissues aged. Unfertilized and recently fertilized carpels did not follow this trend, suggesting different regulatory mechanism of GA signaling in these stages. Characterization of the response to GA of various organs in three seedless cultivars revealed differential response of the berries and rachis. Interestingly, VvDELLA3 transcript levels in the GA-unresponsive berries of cv. Spring blush were significantly higher compared to their levels in the highly responsive berries of cv. Black finger. Assuming that VvDELLA2 and VvDELLA3 are regulating berry size, constructs carrying potential dominant mutations in each gene were created. Furthermore, constitutive silencing of these genes by mIR is underway, to reveal the effect of each gene on the berry phenotype.
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Cohen, Jerry D., and Ephraim Epstein. Metabolism of Auxins during Fruit Development and Ripening. United States Department of Agriculture, August 1995. http://dx.doi.org/10.32747/1995.7573064.bard.
Full textAbstract:
We had proposed to look at several aspects of auxin metabolism in fruit tissues: 1) IAA biosynthesis from tryptophan and IAA biosynthesis via the non-tryptophan pathway; 2) changes in the capacity to form conjugates and catabolites of auxin at different times during fruit development and; 3) the effects of modifying auxin metabolism in fruit tissues. The latter work focused primarily on the maize iaglu gene, with initial studies also using a bacterial gene for hydrolysis of IAA-aspartate. These metabolic and molecular studies were necessary to define potential benefits of auxin metabolism modification and will direct future efforts for crop improvement by genetic methods. An in vitro system was developed for the production of tomato fruit in culture starting from immature flowers in order to ascertain the effect of auxin modification on fruit ripening. IAA supplied to the fruit culture media prior to breaker stage resulted in an increase in the time period between breaker and red-ripe stages from 7 days without additional IAA to 12 days when 10-5 M IAA was added. These results suggest that significant changes in the ripening period could be obtained by alteration of auxin relationships in tomato fruit. We generated transgenic tomato plants that express either the maize iaglu gene or reduced levels of the gene that encodes the enzyme IAA-glucose synthetase. A modified shuttle vector pBI 121 expressing the maize iaglu gene in both sense and antisense orientations under a 35S promoter was used for the study. The sense plants showed total lack of root initiation and development. The antisense transgenic plants, on the other hand, had unusually well developed root systems at early stages in development. Analysis showed that the amount and activity of the endogenous 75 kDa IAGLU protein was reduced in these plants and consequently these plants had reduced levels of IAA-glucose and lower overall esterified IAA.
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Flaishman, Moshe, Herb Aldwinckle, Shulamit Manulis, and Mickael Malnoy. Efficient screening of antibacterial genes by juvenile phase free technology for developing resistance to fire blight in pear and apple trees. United States Department of Agriculture, December 2008. http://dx.doi.org/10.32747/2008.7613881.bard.
Full textAbstract:
Objectives: The original objectives of this project were to: Produce juvenile-free pear and apple plants and examine their sensitivity to E. amylovora; Design novel vectors, for antibacterial proteins and promoters expression, combined with the antisense TFL1 gene, and transformation of Spadona pear in Israel and Galaxy apple in USA. The original objectives were revised from the development of novel vectors with antibacterial proteins combined with the TFL-1 due to the inefficiency of alternative markes initially evaluated in pear, phoshomannose-isomerase and 2-deoxyglucose-6-phosphate phosphatase and the lack of development of double selection system. The objectives of project were revised to focus primarily on the development additional juvenile free systems by the use of another pear variety and manipulation of the FT gene under the control of several promoters. Based on the results creation of fire blight resistance pear variety was developed by the use of the juvenile free transgenic plant. Background: Young tree seedlings are unable to initiate reproductive organs and require a long period of shoot maturation, known as juvenile phase. In pear, juvenile period can last 5-7 years and it causes a major delay in breeding programs. We isolated the TFL1 gene from Spadona pear (PcTFL1-1) and produced transgenic ‘Spadona’ trees silencing the PcTFL1 gene using a RNAi approach. Transgenic tissue culture ‘Spadona’ pear flowered in vitro. As expected, the expression of the endogenous PcTFL1 was suppressed in the transgenic line that showed precocious flowering. Transgenic plants were successfully rooted in the greenhouse and most of the plants flowered after only 4-8 months, whereas the non-transformed control plants have flowered only after 5-6 years of development. Major achievements: Prior to flower induction, transgenic TFL1-RNAi ‘Spadona’ plants developed a few branches and leaves. Flower production in the small trees suppressed the development of the vegetative branches, thus resulting in compact flowering trees. Flowering was initiated in terminal buds, as described for the Arabidopsis tfl1 mutant. Propagation of the transgenic TFL1-RNAi ‘Spadona’ was performed by bud grafting on 'Betulifolia' rootstock and resulted in compact flowering trees. The transgenic flowering grafted plants were grown in the greenhouse under a long photoperiod for one year, and flowered continuously. Pollination of the transgenic flowers with ‘Costia‘ pear pollen generated fruits of regular shape with fertile F1 seeds. The F1 transgenic seedling grown in the greenhouse formed shoots and produced terminal flowers only five months after germination. In addition, grafted F1 transgenic buds flower and fruit continuously, generating hybrid fruits with regular shape, color and taste. Several pear varieties were pollinated with the transgenic TFL1-RNAi ‘Spadona’ pollen including `Herald Harw` that was reported to have resistance to fire blight diseases. The F-1 hybrid seedlings currently grow in our greenhouse. We conclude that the juvenile-free transgenic ‘Spadona’ pear enables the development of a fast breeding method in pear that will enable us to generate a resistance pear to fire blight. Implications: The research supported by this grant has demonstrated the use of transgenic juvenile free technology in pear. The use of the juvenile free technology for enhancement of conventional breeding in fruit tree will serve to enhance fast breeding systems in pear and another fruit trees.
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Firon, Nurit, Prem Chourey, Etan Pressman, Allen Hartwell, and Kenneth J. Boote. Molecular Identification and Characterization of Heat-Stress-Responsive Microgametogenesis Genes in Tomato and Sorghum - A Feasibility Study. United States Department of Agriculture, October 2007. http://dx.doi.org/10.32747/2007.7591741.bard.
Full textAbstract:
Exposure to higher than optimal temperatures - heat-stress (HS) - is becoming increasingly common to all crop plants worldwide. Heat stress coinciding with microgametogenesis, especially during the post-meiotic phase that is marked by starch biosynthesis, is often associated with starch-deficient pollen and male sterility and ultimately, greatly reduced crop yields. The molecular basis for the high sensitivity of developing pollen grains, on one hand, and factors involved in pollen heat-tolerance, on the other, is poorly understood. The long-term goal of this project is to provide a better understanding of the genes that control pollen quality under heat-stress conditions. The specific objectives of this project were: (1) Determination of the threshold heat stress temperature(s) that affects tomato and sorghum pollen quality whether: a) Chronic mild heat stress conditions (CMHS), or b) Acute heat stress (AHS). (2) Isolation of heat-responsive, microgametogenesis-specific sequences. During our one-year feasibility project, we have accomplished the proposed objectives as follows: Objectrive 1: We have determined the threshold HS conditions in tomato and sorghum. This was essential for achieving the 2nd objective, since our accumulated experience (both Israeli and US labs) indicate that when temperature is raised too high above "threshold HS levels" it may cause massive death of the developing pollen grains. Above-threshold conditions have additional major disadvantages including the "noise" caused by induced expression of genes involved in cell death and masking of the differences between heatsensitive and heat-tolerant pollen grains. Two different types of HS conditions were determined: a) Season-long CMHS conditions: 32/26°C day/night temperatures confirmed in tomato and 36/26°C day maximum/night minimum temperatures in sorghum. b) Short-term AHS: In tomato, 2 hour exposure to 42-45°C (at 7 to 3 days before anthesis) followed by transfer to 28/22±2oC day/night temperatures until flower opening and pollen maturation, caused 50% reduced germinating pollen in the heat-sensitive 3017 cv.. In sorghum, 36/26°C day/night temperatures 10 to 5 days prior to panicle emergence, occurring at 35 days after sowing (DAS) in cv. DeKalb28E, produced starch-deficient and sterile pollen. Objective 2: We have established protocols for the high throughput transcriptomic approach, cDNA-AFLP, for identifying and isolating genes exhibiting differential expression in developing microspores exposed to either ambient or HS conditions and created a databank of HS-responsivemicrogametogenesis-expressed genes. A subset of differentially displayed Transcript-Derived Fragments (TDFs) that were cloned and sequenced (35 & 23 TDFs in tomato and sorghum, respectively) show close sequence similarities with metabolic genes, genes involved in regulation of carbohydrate metabolism, genes implicated in thermotolerance (heat shock proteins), genes involved in long chain fatty acids elongation, genes involved in proteolysis, in oxidation-reduction, vesicle-mediated transport, cell division and transcription factors. T-DNA-tagged Arabidopsis mutants for part of these genes were obtained to be used for their functional analysis. These studies are planned for a continuation project. Following functional analyses of these genes under HS – a valuable resource of genes, engaged in the HS-response of developing pollen grains, that could be modulated for the improvement of pollen quality under HS in both dicots and monocots and/or used to look for natural variability of such genes for selecting heat-tolerant germplasm - is expected.