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Development of single molecule-sensitive, imaging probes targeting native RNALifland, Aaron William 26 June 2012 (has links)
The localization, trafficking and regulation of messenger ribonucleic acids (RNA) and viral RNA play crucial roles in cellular homeostasis and disease pathogenesis. In recent years biochemical and molecular biology methods used to study RNA function have made several important advances in the areas of RNA interference, expression of transgenes, and the sequencing of transcriptomes. In contrast, current technologies for imaging RNA in live cells remain in limited use.
Previous studies of RNA localization and dynamics have relied primarily on the expression of a reporter RNA and a fluorescent protein fusion that binds to aptamer sequences in the expressed RNA. While these plasmid based systems offer methodological flexibility, there remains a need to develop methods to image native, non-engineered RNA as plasmid derived RNAs may not have the same regulatory elements (3'UTR and introns) or copy number as the native RNA. Additionally, viral pathogenesis is often sensitive to the size and sequence of their genomic RNA and may not be suitable for study using engineered systems.
We sought to develop and validate a new method for imaging native, non-engineered RNA with single molecule-sensitivity. These probes have four important properties. They are modular, compatible with fixation and immunostaining, bind quickly and specifically to targets, and do not interfere with RNA function. We built upon the technique of delivering exogenous, linear probes that bind to their target by Watson-Crick base pairing. The probes are multiply labeled and tetramerized to increase their brightness. To validate the probes, targeting and utility was demonstrated in two model systems: beta-actin mRNA to show targeting of an endogenous target and the genomic RNA of human respiratory syncytial virus to show targeting of a viral RNA target. All video files are in QuickTime format.
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Localisation signals within the c-myc and c-fos 3'untranslated regionsDalgleish, Gillian Denise January 2000 (has links)
No description available.
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Regulation of cytokine mRNA stabilty / Cheryl Yvette Brown.Brown, Cheryl Yvette January 1996 (has links)
Copies of author's previously published works inserted. / Includes bibliographies. / 1 v. : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology and Div. of Human Immunology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, 1996
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Chicken globin mRNA and its precursor / by Robert John CrawfordCrawford, Robert John January 1977 (has links)
Typescript (photocopy) / vii, 98 leaves : ill. ; 28 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1977
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Chicken histone H5 mRNA and its genesScott, Andrew Charles January 1975 (has links)
1. The work described in this thesis forms part of an investigation of eukaryotic gene control. The system studied was the avian erythroid cell series since it is possible to isolate pure populations of the various cell types which have well-defined biochemical activities. These cells contain an unusual tissue - specific histone H5, which may be involved in the progressive repression of transcription observed as these cells differentiate. Although the gene controlling function of this histone must be at a very gross level, this represents a unique opportunity to investigate one facet of gene control. Probably the most sensitive technique is to assay for specific messenger RNA and gene sequences by hybridisation to an appropriate probe. The aim of this thesis was to prepare such a probe from H5 mRNA and to use it to calculate the reiteration frequency of the H5 gene in the chicken genome. 2. The cells employed were chicken reticulocytes since the only histone made in these cells is H5. Experiments were conducted which demonstrated that H5 mRNA is probably a minor species compared to globin mRNA in these cells. Furthermore, calculations indicate that the two mRNAs are probably of similar molecular weight which may complicate the isolation of H5 mRNA. As a result globin mRNA was first purified and characterised. Properties which may have proved useful in the separation of this mRNA from H5 mRNA are discussed. The globin mRNA was used to optimise techniques for the in vitro translation and identification of chicken mRNAs. This was considered necessary as mRNAs from different sources vary in the conditions required for optimal translation and it was reasoned that mRNAs from the same cell would have similar optima. 3. Total polysomal RNA was fractionated on the basis of size and poly A content. Although large amounts of globin mRNA were present, H5 mRNA could only be detected in the non - poly A containing RNA. Even in this fraction however, there was still a large excess of globin mRNA which was difficult to remove due to the demonstrated similarity of their molecular weights. 4. Since it had proved impossible to isolate the H5 mRNA by conventional techniques, immunological methods of isolating the polysomes producing H5 were investigated. Using immunoadsorbents, mRNA was prepared in small amounts which programmed the synthesis in vitro of more than 70 % H5. The yield and specificity were improved by modifying the procedure to indirect immunoprecipitation followed by oligo ( dT ) - cellulose chromatography. The resulting mRNA programmes the synthesis in vitro of more than 90 % H5. The chemical purity of the mRNA is discussed. 5. The immunologically prepared H5 mRNA was not copied into cDNA by RNA - dependent DNA - polymerase. Since this was probably due to the lack of a 3 ' poly A tract on the mRNA, an enzyme was purified and characterised which would add such a tract. The enzymically modified mRNA could then be copied into cDNA of high specific activity. 6. The H5 cDNA was characterised in terms of size and fidelity of copying. By hybridisation analysis it was demonstrated that the amount of contaminating rRNA and globin mRNA complementary sequences present in the cDNA was insignificant. The complexity of the cDNA was shown to be of the same size as the H5 mRNA and will back hybridise to this mRNA to greater than 75 %. These results are discussed to demonstrate that the cDNA is a faithful copy of H5 mRNA. The possible uses of the resulting probe are also discussed. 7. The H5 cDNA was employed to quantify the number of H5 genes in the chicken genome. The significance of this result is discussed in terms of the known reiteration and organisation of histone genes in other species, and the possible role of H5 as a gene control agent. / Thesis (Ph.D.)--Department of Biochemistry, 1975.
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High-resolution studies of mRNA expression in brain : a search for genes differently expressed in schizophrenia /Castensson, Anja, January 2003 (has links)
Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.
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Preproenkephalin gene and mRNA : studies of structure, function, cocaine responses in an animal model, and genetic association with human opiate addiction /LaForge, Karl Steven, January 2004 (has links)
Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.
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Characterization of non-coding mRNA in Epstein-Barr virus /Isaksson, Åsa, January 2007 (has links)
Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2007. / Härtill 4 uppsatser.
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Tolerance and antagonism to allopregnanolone effects in the rat CNS /Türkmen, Şahruh, January 2006 (has links)
Diss. (sammanfattning) Umeå : Univ., 2006. / Härtill 4 uppsatser.
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Functional elucidation of BS69 /Zhang, Wei. January 2008 (has links)
Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 91-108). Also available in electronic version.
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