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1
Ward, Karen. "Margins and the frame of reading." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq24940.pdf.
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SPITALI, Pietro. "ANTISENSE MEDIATED DYSTROPHIN READING FRAME RESTORATION." Doctoral thesis, Università degli studi di Ferrara, 2010. http://hdl.handle.net/11392/2389323.
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Exon skipping using antisense oligonucleotides (AONs) has successfully been used to
reframe the mRNA in various DMD (Duchenne muscular dystrophy) patients carrying
deletions and in the mdx mouse model. This study can be devided in two parts: in the
first part we have tested the feasibility of the exon skipping approach for patients with
small mutations in in-frame exons, while in the second part a quantitative comparison of
exon skipping revealing techniques is addressed.
We first identified 55 novel disease-causing point mutations. We selected 5 patients
with nonsense or frameshifting mutations in exons 10, 16, 26, 33 and 34. Wild type and
mutation specific 2‟OMePS AONs were tested in cell-free splicing assays and in
cultured cells derived from the selected patients. The results obtained confirm cell-free
splicing assay as an alternative system to test exon skipping propensity when patients‟
cells are unavailable. In myogenic cells, similar levels of exon skipping were observed
for wild type and mutation specific AONs for exons 16, 26 and 33, while for exon 10 and
exon 34 the efficiency of the AONs was significantly different. Interestingly, in some
cases skipping efficiencies for mutated exons were quite dissimilar compared to what
previously reported for the respective wild type exons. This behaviour may be related to
effect of the mutations on exon skipping propensity and highlights the complexity of
identifying optimal AONs for skipping exons with small mutations.
In the second part we compared different techniques to reveal the exon skipping levels
in the muscles of 7 different mdx mice. An absolute quantification of the dystrophin
transcript amount was possible using a digital array. Results underline the low
expression of the dytrophin gene and the amount needed to correctly quantify the exon
skipping percentage.
3
Lindström, Mikael. "Functional characterization of the alternative reading frame protein p14ARF /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-917-x.
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Amin, Unum. "Cellular characterisation of small Open Reading Frame function in Drosophila melanogaster." Thesis, University of Sussex, 2016. http://sro.sussex.ac.uk/id/eprint/65080/.
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As our knowledge of the genome expands, so does our understanding of the characteristics of what we define as genes. Small Open Reading Frame (smORF) genes have eluded gene annotation until very recently, and evidence is mounting that these very short nucleotide sequences encode functional peptides that are ≤100 amino acids in size. From work conducted in the fruit fly, our lab has successfully characterised three Drosophila smORFs, of which two have been shown to have a function in higher vertebrates, including humans. The functional characterisation of one of these conserved smORF encoded peptides (SEPs), Hemotin, is presented in this thesis. Though the overall number is still low compared to the abundance of potential smORF-encoding genes in Drosophila, the information gathered here allows us to speculate on the wider role of smORF peptides through cell-based imaging studies conducted on Drosophila cells. Here, I will discuss the various techniques that can and should be employed in order to study the functions of SEPs. Chapter III describes the various phenotypic studies conducted on the Hemotin smORF which is expressed in Drosophila haemocytes, and are integral to the fruit fly immune system. This study showed that connecting subcellular localisation of an SEP to a direct functional assay in cells can reveal functional characteristics of the peptide for further study. Chapter IV details the results from a tagging-transfection assay, which began initially as a way to independently corroborate the translation of smORF mRNAs that were assessed as such by Ribosome Profiling. This experiment resulted in the discovery of several mitochondrial-localised SEPs in Drosophila S2 cells, opening the door for the direct functional assay described in Chapter V. The results from a small-scale RNAi screen conducted on the mitochondria of S2 cells provided a reliable read-out for functionality of a large proportion of the smORFs that were screened. This assay can potentially be used as a phenotypic read-out of mitochondrial-SEP function in any cell or tissue type. Elucidation of smORFs and the functions of the peptides that they encode will help us to expand the Drosophila proteome, along with providing evidence of their functionality across every organism in which they are found. Considering that characterised SEPs play very important roles in physiology and health, it is time for smORFs to be acknowledged as the important genomic elements that they are.
5
Forrest, Megan E. "Regulation of Mammalian Messenger RNA Stability via the Open Reading Frame." Case Western Reserve University School of Graduate Studies / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1579862741902687.
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Nerlick, Stephen Tyler. "Inhibition of Xnos1 Translation by Structural Elements in the Open Reading Frame." Scholarly Repository, 2008. http://scholarlyrepository.miami.edu/oa_theses/141.
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The spatio-temporal regulation of translation is critical to the proper development of all organisms. The presence of Translational Control Elements (TCEs) in Un-Translated Regions (UTRs) is one feature common to all regulated eukaryotic mRNAs examined to date. These TCEs serve as binding sites for sequence specific proteins or small regulatory RNAs that recruit other accessory proteins that inhibit translation. Xnos1, a localized RNA in the germ plasm of Xenopus, is negatively regulated by an unknown mechanism. We used in vivo and in vitro translation assays, competition assays, and ribosome binding assays in order to determine the location of the Xnos1 TCE and its mechanism of repression. The Xnos1 TCE is located in the open reading frame, a departure from the canonical translational regulation mechanisms. This TCE is predicted to form conserved secondary structures in the first 75 nucleotides of the open reading frame (ORF). In vitro translation assays demonstrated that the repression can be partially relieved by either denaturing the transcripts or by introducing point mutations that weaken the secondary structure. This TCE cannot be relieved by competition and therefore is likely not to require the presence of a repressor protein. The structural regulation of translation by a TCE in the open reading frame is a novel mechanism of repression for a eukaryotic mRNA.
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Mumtaz, Muhammad Ali Shahzad. "High-throughput assessment of small open reading frame translation in Drosophila melanogaster." Thesis, University of Sussex, 2016. http://sro.sussex.ac.uk/id/eprint/65096/.
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Hundreds of thousands of putative small ORFs (smORFs) sequences are present in eukaryotic genomes, potentially coding for peptides less than 100 amino acids. smORFs have been deemed non-coding on the basis of their high numbers and their small size that makes it extremely challenging to assess their functionality both bioinformatically and biochemically. The recently developed Ribo-Seq technique, which is the deep sequencing of ribosome footprints, has generated significant controversy by showing extensive translation of smORFs outside of annotated protein coding regions, including putative non-coding RNAs.. Our lab adapted the Ribo-Seq technique by combining it with the polysome fractionation in order to assess smORF translation in Drosophila S2 cells. This thesis provides a high-throughput assessment of smORF translation in Drosophila melanogaster by firstly implementing complementary techniques such as transfection-tagging and Mass spectrometry methods in order to provide an independent corroboration of the S2 cell data (Chapter 3). Secondly, the in order to expand the catalogue of smORFs that are translated, I significantly improve upon the yield and sequencing efficiency of the Poly-Ribo-Seq protocol while adapting it to Drosophila embryos and then implementing it across embryogenesis divided in to Early, Mid and Late stages (Chapter 4). Currently, there is still a lot of debate in the field with regards to Ribo-Seq data analysis, and various computational metrics have been developed aimed at discerning 'real' translation events to background noise. Chapter 5 explores some of the metrics developed and establishes a translation cut-off suitable for designating small ORFs as translated. Altogether, the improvements introduced to the protocol and my data analysis shows the translation of 500 annotated smORFs, 500 smORFs in long non-coding RNAs and 5,000 uORFs, of which only one-third of each type of smORF has previous evidence of translation. These findings strengthen the establishment of smORFs as a distinct class of genes that significantly expand the protein coding complement of the genome.
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Chavez, Elisabeth [Verfasser]. "Reading Beyond the Diaspora : A Responsible Reading of Recent North American Fiction Outside the Ethnicity Frame / Elisabeth Chavez." Berlin : Freie Universität Berlin, 2021. http://d-nb.info/1229917292/34.
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Ren, Qian. "Characterization of alternative reading frame selection by a viral internal ribosome entry site." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/48576.
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Dicistroviruses possess a positive-sense, monopartite single-strand RNA genome
that encodes two open reading frames containing the nonstructural and structural
polyproteins (ORF1 and ORF2) separated by the intergenic region (IGR) internal ribosome
entry site (IRES). Translation of each ORF is directed by distinct IRESs, a 5’ untranslated
region (UTR) and an IGR IRES. Previous bioinformatic studies have shown that a subset of
dicistroviruses contain an overlapping gene in the +1 translational reading frame within the
structural polyprotein gene near the IGR IRES region. We hypothesize that the IGR IRES
directs translation of two overlapping ORFs, a novel +1 frame ORFx and the 0 frame ORF
which encodes the viral structural polyprotein. In this thesis, using Israeli acute paralysis
virus (IAPV) as a model, the existence and start site of ORFx were identified using
mutagenesis and Mass Spectrometry analyses. In addition, the structural elements within
the IAPV IGR IRES that determine alternative reading frame translation initiation were
explored. Lastly, the localization of overexpressed tagged-ORFx in Drosophila S2 cells
was examined to gain insights of its function. Summarizing, we have discovered a novel
mechanism that increases the coding capacity of a virus through an IGR IRES. These
studies of IAPV IGR-IRES will further our understanding of IRESs mediated translation
initiation and reading frame decoding.Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate
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Yates, Paula Rachel. "A functional characterisation of the African swine fever virus open reading frame k9L." Thesis, University of Liverpool, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283497.
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Watts, Talina Christensen. "Genetic Analysis of the Role of SmpB in Establishing the Reading Frame on tmRNA." BYU ScholarsArchive, 2008. https://scholarsarchive.byu.edu/etd/1728.
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Ribosomes translate the genetic information encoded by mRNA into proteins. Defective mRNAs can cause stalling of translating ribosomes. The molecule tmRNA (transfer-messenger RNA) rescues stalled ribosomes in eubacteria. Together with its protein partner SmpB, tmRNA mimics a tRNA by entering the ribosomal A site and linking an alanine residue to the growing polypeptide chain. The ribosome then abandons the defective mRNA template and resumes translation on tmRNA, adding ten more amino acids to the nascent polypeptide. As a result of tmRNA action, stalled ribosomes are released and recycled, the defective mRNA is destroyed, and the aborted protein product is tagged for destruction by proteases. It is unknown how the ribosome correctly chooses the position on tmRNA to resume translation. Previous studies implicate the sequence UAGUC found immediately upstream of the first codon in the tmRNA open reading frame. These nucleotides are highly conserved in natural tmRNA sequences. Mutations in this area cause loss of tmRNA function and improper frame choice. Using a genetic selection that ties the life of E. coli cells to the function of tmRNA, we have identified several SmpB mutants that rescue an inactive tmRNA in which this upstream sequence was altered. This links SmpB to the function of these key tmRNA nucleotides. We show that our SmpB mutants affect frame choice using an in vivo assay for tagging in the various frames. We conclude that SmpB plays a role in setting the reading frame on tmRNA.
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Au, Hilda Hiu Tung. "Elucidating the mechanism of reading frame selection by a viral internal ribosome entry site." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/60232.
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The Dicistroviridae intergenic region internal ribosome entry site (IGR IRES) exhibits the remarkable ability to bind the conserved core of the ribosome with high affinity. By mimicking the conformation of a tRNA, the IGR IRES can bypass the requirement for canonical initiation factors and Met-tRNAi, and initiate translation from a non-AUG start codon in the ribosomal A site. The pseudoknot (PKI) domain of the IRES engages the decoding center upon initial ribosome binding, and subsequently translocates into the P site to allow delivery of the incoming aminoacyl-tRNA. Within the P site, the IRES adopts a conformation that is reminiscent of a P/E hybrid state tRNA to effectively co-opt the canonical elongation cycle. How the IGR IRES establishes the translational reading frame in the absence of initiation factors remains an outstanding question. Here, we elucidate the mechanism of reading frame selection by performing mutagenesis and biochemical assays to explore the function of specific IRES structural elements. We demonstrate that constituents of the Cricket paralysis virus PKI domain, including the helical stem, anticodon:codon-like base-pairing, and the variable loop region are optimized for IRES-mediated translation. Additionally, we reveal through extensive structural and biochemical studies that stem-loop III of the Israeli acute paralysis virus (IAPV) IRES mimics the acceptor stem of tRNA and functions in supporting efficient 0 frame translation. Finally, we established an infectious chimeric clone to investigate how translational regulation by the IAPV IRES affects the viral life cycle. Studies using this chimera demonstrate that formation of stem-loop VI upstream of the IAPV IRES contributes to optimal IRES activity and viral yield. Our findings suggest that extensive and complete tRNA-mimicry by the IAPV IGR IRES facilitates IRES-mediated translation and reading frame selection.Medicine, Faculty of
Graduate
13
Näsvall, Joakim. "Wobble modifications and other features in transfer RNA important for decoding and reading frame maintenance." Doctoral thesis, Umeå universitet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1399.
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Transfer RNA (tRNA) is the adaptor molecule responsible for bringing the correct amino acid to the ribosome during protein synthesis. tRNA contains a number of modified nucleosides, which are derivatives of the four normal nucleosides. A great variety of modifications are found in the anticodon loop, especially at the first (wobble) position of the anticodon. According to Crick’s wobble hypothesis, a uridine at the wobble position of tRNA recognize codons ending with A and G. Uridine-5-oxyacetic acid (cmo5U34), found at the wobble position of six species of tRNA in Salmonella enterica, have been predicted to expand the codon recognition of uridine to include U-ending, but not C-ending codons. To study the function of cmo5U34 we have identified two genes, cmoA and cmoB, which are required for the synthesis of cmo5U34 in tRNA. We have shown that the proline, alanine and valine tRNAs containing cmo5U34 are capable of reading codons ending with any of the four nucleotides, while the threonine tRNA is not, and the importance of having cmo5U is different for the different tRNAs. In addition, we found that cmo5U is important for efficient reading of G-ending codons, which is surprising considering the wobble hypothesis, which states that uridine should read G-ending codons. The dominant +1 frameshift suppressor sufY suppresses the hisC3737 +1 frameshift mutation. We have demonstrated that sufY induces frameshifting at CCC-CAA (Pro-Gln), when tRNAPro[cmo5UGG] occupies the P-site. sufY mutants accumulate novel modified nucleosides at the wobble position of tRNAs that should normally have (c)mnm5s2U34. The presence of an extra sidechain (C10H17) on the wobble nucleoside of tRNAGln[(c)mnm5s2U] leads to slow decoding of CAA codons, inducing a translational pause that allows the P-site peptidyl-tRNAPro[cmo5UGG] to slip into the +1 frame. We have characterized 108 independent frameshift suppressor mutants in the gene encoding tRNAPro[cmo5UGG]. The altered tRNAs are still able to read all four proline codons in the A-site, but induce frameshifts after translocation into the P-site. Some of the mutations are in regions of the tRNA that are involved in interactions with components of the P-site. We hypothesize that the ribosomal P-site keeps a “grip” of the peptidyl-tRNA to prevent loss of the reading frame.
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Elder, Kenneth James. "The characterization of an open reading frame involved in clavulanic acid biosynthesis in Streptomyces clavuligerus." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ34357.pdf.
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Näsvall, Joakim. "Wobble modifications and other features in transfer RNA important for decoding and reading frame maintenance /." Umeå : Department of Molecular Biology, Umeå University, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1399.
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Magny, Emile Gerard. "Functional characterisation of pncr003;2L, a small open reading frame gene conserved from Drosophila to humans." Thesis, University of Sussex, 2014. http://sro.sussex.ac.uk/id/eprint/48976/.
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Small open reading frame genes (smORFs) are a new class of genes, which emerged from the revision of the idea that open reading frames have to be longer than 100 codons to be protein coding and functional. Although bio-informatics evidence suggests that thousands of smORF genes could exist in any given genome, proof of their functional relevance can only be obtained through their functional characterization. This work represents such a study for a Drosophila smORF (pncr003;2L), which was initially misannotated as a non-coding RNA because of its lack of a canonical long open reading frame. Here I show that pncr003;2L codes for two small peptides of 28 and 29 aa, expressed in somatic and cardiac muscles. After generating a null condition for this gene, I use the adult Drosophila heart as a system to assess the function of pncr003;2L. With this system, I show that the small pncr003;2L peptides regulate heart contractions by modulating Ca2+ cycling in cardiac muscles, with either lack or excess of function of these peptides leading to cardiac arrhythmias, and abnormal calcium dynamics. Finally, through an extensive homology study, I show that these small peptides share a great amount of structural and functional homology with the peptides encoded by the vertebrate smORFs sarcolipin (sln) and phospoholamban (pln), which act as major regulators of the Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA), the channel responsible for calcium uptake into the ER following muscle contraction. These results highlight the importance of the pncr003;2L smORF and the Drosophila system, for the study of cardiac pathologies, but most importantly, they show that this family of peptides, conserved across evolution, represent an ancient system for the regulation of calcium trafficking in muscles. This work corroborates the prevalence, and relevance of this novel class of genes, and shows that closer attention should be given to smORFs in order to determine the full extent of their biological contribution.
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Alberto, Fernando Lopes. "Avaliação do transcriptoma da leucemia mieloide cronica por ORESTES (Open Reading Frame Expression Sequence Tags)." [s.n.], 2003. http://repositorio.unicamp.br/jspui/handle/REPOSIP/309337.
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Orientador: Fernando Ferreira CostaTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-08T16:15:41Z (GMT). No. of bitstreams: 1 Alberto_FernandoLopes_D.pdf: 5633645 bytes, checksum: 7354434461195623d47c6efe24126229 (MD5) Previous issue date: 2003
Resumo: Câncer é o resultado de anomalias no genoma acumuladas ao longo do tempo e corresponde a doença genética mais freqüente na população humana. Os últimos anos têm presenciado enormes avanços tecnológicos que facilitaram o desenvolvimento de técnicas para análise em larga escala do conjunto dos genes expressos nos diferentes tecidos, sendo responsável pela explosão do conhecimento em medicina molecular. No presente trabalho avaliamos o transcriptoma da leucemia mielóide crônica através da técnica de ORESTES (Open Reading Frame Expressed Sequence Tags). As seqüências geradas foram agrupadas de acordo com sua homologia através do pacote de bioinformática Phrap. Essa estratégia aumentou o grau de cobertura genômica, já que as seqüências originais apresentavam tamanho máximo de 500 a 700 bases. Após a retirada dos contaminantes, obtivemos 2933 agrupamentos de seqüências (contigs) e 11568 seqüências isoladas. Cada contig representa um gene em potencial e o seu conjunto foi submetido a comparação com o banco de dados público nr para possibilitar a anotação e classificação funcional. Os agrupamentos ORESTES foram classificados em comunicação celular (19%); crescimento, desenvolvimento e manutenção celular (9%); metabolismo celular (30%); estrutura, motilidade e transporte celular (15%); função desconhecida (28%). Adicionalmente, estabelecemos o mapeamento cromossômico dos contigs obtidos, observando distribuição relativamente homogênea e proporcional representada em todos os cromossomos humanos. O mapeamento dos contigs gerados por ORESTES apresentou-se com padrão de distribuição semelhante ao observado por Venter e colaborasores, 2001. Comparamos o padrão de expressão obtido com o transcriptoma de medula óssea de paciente portador de anemia hemolítica hereditária (doença hematológica não neoplásica) gerado por SAGE (Serial Analysis of Gene Expression). Os genes encontrados foram ordenados de acordo com sua representatividade ou nível de expressão. Não houve correlação entre os padrões de expressão observados pos SAGE e por ORESTES. Do conjunto dos 100 genes mais representados nos ORESTES, dez foram selecionados para validação da expressão por PCR quantitativo em tempo real, utilizando o agente fluorescente SybrGreen I. Identificamos e confirmamos o aumento da expressão em nove genes na LMC. Destes, sete tinham aumento superior a 10 vezes o controle (CAMP, ABR, DEFA3, CSF3R, MLL2, S100A8 e MS4A3), com dois deles (DEFA3 e ABR) com aumento de cerca de 50 vezes ou mais. A análise funcional particularizada para cada um deste genes em LMC tem o potencial de identicar possíveis alvos para novas intervenções terapeuticas. O método mostrou-se poderoso para evidenciar e validar as diferenças de expressão gênica observadas pelas técnicas de seqüenciamento utilizadas anteriormente. Os dados deste trabalho constituem a primeira descrição do transcriptoma da leucemia mielóide crônica
Abstract: The complete collection of transcripts generated from the human genome cannot be predicted from the genome sequence, but should be directly determined for each tissue, due to variations of gene expression in different tissues and disease states, and because genes can encode multiple transcripts derived from alternate splicing and polyadenylation sites. As part of larger project that produced up to 1.2 million ORESTES (open reading frames expressed sequence tags) from different cancer tissues, we constructed a set of cDNAs obtained from bone marrow cells of patients with CML, that represented partial expressed gene sequences that are biased toward the central coding regions of the resulting transcripts (Dias-Neto E et al, Proc Nat Acad Sci USA 97:3491, 2000). The 27,361 ESTs were assembled into contigs using the Phrap package producing 2933 clusters containing 2 to 210 ESTs (leaving 11568 isolated sequences). Functional categories were attributed to the contigs on the basis of the annotation of the matched sequences in nr, into 5 major functional categories and 22 subcategories (cell communication (19%); cell growth, development and m contigs formed by the larger number of EST in bone marrow cells was co mpared with a control bone marrow sample obtained from a non-neoplastic hematologic condition (hereditary hemolytic anemia) and evaluated with SAGE (serial analysis of gene expression). Ten out of 100 most representative contigs had their expression data evaluated further by realtime quantitative PCR with Sybr Green I reagent, confirming the ORESTES data. Seven out of the 10 genes were more than 10-fold upregulated (CAMP, ABR, DEFA3, CSF3R, MLL2, S100A8 e MS4A3) and two additional genes were 50-fold or higher upregulated in CML (DEFA3 e ABR). The wealthy of information provided by this approach demonstrates its usefulness for the analysis of gene expression in specific hematopoietic tissues and diseases
Doutorado
Clinica Medica
Doutor em Clínica Médica
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McFadden, Nora Ann. "Identification and characterisation of an overlapping open reading frame (ORF4) within the murine norovirus genome." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/10724.
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Caliciviruses are single-stranded positive-sense RNA viruses, most commonly associated with outbreaks of gastroenteritis in humans. In addition to the three open reading frames (ORFs) typical of caliciviruses a highly conserved fourth overlapping ORF within the murine norovirus (MNV) genome was identified and characterised during this project. ORF4 overlaps and is contained within the capsid encoding ORF2 in a +1 frame. Once a suitable antibody had been generated, immunoblotting was used to show that the ORF4 protein is produced during MNV infection. Although ORF4 mutant viruses were viable and replicated to wild-type MNV levels in tissue culture, pressure to restore ORF4 expression upon serial passage under specific conditions was demonstrated. Importantly, the ORF4 knockout virus was significantly attenuated in STAT1-/- mice. Proteomic analysis and a commercial yeast two-hybrid screen were used to identify host cell factors which interact with the ORF4 protein and confocal imaging was employed to examine cellular localisation. These approaches indicated that the ORF4 protein localises to the mitochondria and in vitro assays were subsequently used to demonstrate an involvement of the ORF4 protein in MNV induced apoptosis. As cells infected with the knockout virus showed an earlier and higher degree of apoptosis induction compared to wild-type infected cells, it is possible that the ORF4 protein may function to delay the onset of apoptosis during MNV infection. Whether or not the ORF4 protein has antiapoptotic activity or whether the difference in apoptotic phenotype is an indirect consequence of ORF4 protein function remains to be investigated. These data indicate that the ORF4 protein represents a novel, previously uncharacterised virulence determinant in MNV.
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Ruan, Hangjun. "Mechanisms of translational regulation of S-adenosylmethionine decarboxylase mediated by the upstream open reading frame /." Thesis, Connect to this title online; UW restricted, 1996. http://hdl.handle.net/1773/9243.
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Márquez, Viter. "Switching off the mechanism for maintaining the Ribosomal reading frame: translational regulation of release factor 2." [S.l.] : [s.n.], 2003. http://www.diss.fu-berlin.de/2003/223/index.html.
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Hung, Wing-ki. "Functional study of BamH1 a rightward open reading frame 1 (BARF1) expression in nasopharyngeal epithelial cells /." View the Table of Contents & Abstract, 2006. http://sunzi.lib.hku.hk/hkuto/record/B37133378.
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Hung, Wing-ki, and 孔穎祺. "Functional study of BamH1 a rightward open reading frame 1 (BARF1) expression in nasopharyngeal epithelial cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B45010717.
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Nolan, Lesley A. "The Epstein-Barr virus (EBV) open reading frame BDLF3 codes for a 100-150 kilodalton glycoprotein." Thesis, University of Bristol, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294395.
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Martins, Camila Augusta de Oliveira. "Seqüências ORESTES (open reading frame expressed sequence tags) de trypanosoma cruzi e transcrição de DNA satélite." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-19052008-135857/.
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Nos bancos de dados de T. cruzi há cerca de 3.750 ESTs de amastigotas e tripomastigotas, seqüenciadas a partir das extremidades 5\' ou 3\' de clones de cDNA. A metodologia ORESTES (Open Reading Frame Expressed Sequence Tags) possibilita obter seqüências transcritas parciais derivadas majoritariamente da porção central dos mRNAs, favorecendo a descoberta de novos genes. Neste trabalho, caracterizamos ORESTES de formas infectantes amastigotas e tripomastigotas da cepa humana VL10 (ATVL). A metodologia foi padronizada com formas epimastigotas da cepa CL Brener (ECL), monitorando-se nas preparações de mRNA a contaminação por DNA e a integridade dos transcritos. Populações de cDNA foram obtidas utilizando-se diferentes iniciadores aleatórios. O mesmo iniciador foi empregado nas etapas de RT e PCR, realizada em condições de baixa estringência. Obtivemos 776 e 1522 ORESTES de ECL e ATVL, respectivamente. Após análise com o programa PHRED, aceitaram-se 745 ORESTES de ECL e 1476 de ATVL. As ORESTES apresentaram um tamanho médio de 680 pb e um conteúdo de G+C de 53%. O agrupamento com CAP3 gerou 463 agrupamentos de ECL (360 singletons e 103 contigs) e 454 de ATVL (337 singletons e 117 contigs). A anotação foi feita utilizando-se o programa BLAST contra o banco nr do NCBI. Na biblioteca de ATVL observamos um número elevado de seqüências de RNA ribossômico (27%), amplificadas preferencialmente por dois iniciadores. Para ECL, a contaminação por rRNA foi de 3,6%. Para cerca de 50% das ORESTES de ATVL (n= 729) foi encontrada similaridade em bancos de dados de proteínas. Destas, 316 apresentaram similaridade com proteínas putativas conhecidas e 413 foram anotadas como proteínas hipotéticas e hipotéticas conservadas. Para 87 ORESTES de ATVL (5,9%) não foi observada nenhuma similaridade. 628 ORESTES de ECL (84%) apresentaram similaridade com proteínas depositadas em bancos públicos, ao passo que nenhuma similaridade foi encontrada para 18 cDNAs (2,4%). Ensaios de Southern blot confirmaram a presença de 4 ORESTES no match analisadas nos genomas das duas cepas. Não puderam ser atribuídas a processos biológicos conhecidos 39% e 68% das seqüências dos contigs de ECL e ATVL, respectivamente. Nos processos de Proteólise e Peptidólise, estão incluídas 11% das ORESTES de ECL e 0,3% das ORESTES de ATVL. Outras diferenças funcionais putativas foram observadas. A abundância diferencial dos transcritos de algumas ORESTES foi analisada por northern blot nos estágios evolutivos das cepas. Southern blot do contig ATVL95 originou um padrão de hibridização com múltiplas bandas, característico de seqüências repetitivas. Este contig corresponde ao transcrito do DNA satélite de 195 pb (195 SAT), uma seqüência repetitiva que perfaz cerca de 10% do genoma de T. cruzi e cuja transcrição é controversa na literatura. A transcrição do 195 SAT foi comprovada por experimentos de + northern blot e por RT-PCR. Transcritos de 195 SAT foram detectados nas frações de RNA de poliA+ e poliA-. Esses transcritos não conteriam a seqüência SL, presente nos mRNAs de tripanossomatídios. e poliA Utilizando oligonucleotídios complementares às duas fitas de 195 SAT concluímos que ambas são transcritas. Embora esteja claro que 195 SAT é transcrito, sua função biológica permanece desconhecida.In T. cruzi databases, aproximately 3.750 ESTs of amastigotes and trypomastigotes, sequenced from the 3´ or 5´ ends cDNA clones can be found in T. cruzi databases. The ORESTES (Open Reading Frame Expressed Sequence Tags) methodology generates partial transcribed sequences derived mainly from the central portions of mRNAs, favoring the discovery of new genes. In this work, we have characterized ORESTES sequences from the infective amastigote and trypomastigote forms of the human strain VL10 (ATVL). The methodology was standardized with epimastigotes of the CL Brener strain (ECL), monitoring in the mRNA population DNA contamination and the integrity of the transcripts. cDNA populations were obtained using different arbitrarily selected, nondegenerate primers. The same primer was used in the RT and PCR steps, performed under low-stringency conditions. We obtained 776 and 1522 ORESTES of ECL and ATVL, respectively. After analysis with PHRED program, 745 ORESTES of ECL and 1476 of ATVL were accepted for further characterization. ORESTES showed a medium size of 680 bp and a G+C content of 53%. Clustering with CAP3 generated 463 unique sequences of ECL (360 singletons and 103 contigs) and 454 of ATVL (337 singletons and 117 contigs). The annotation was made with BLAST program against the NCBI nr database. In the ATVL library we observed an elevated number of ribosomal RNA sequences (27%), amplified mainly by two primers. In the ECL library, rRNA contamination was about 3.6%. Approximately 50% of the ATVL sequences (n= 729) were found in protein databases. From these, 316 showed similarity with putative known proteins and 413 were annotated as hypothetical proteins and hypothetical conserved proteins. No hit was observed for 87 ORESTES of ATVL (5.9%). 628 ORESTES of ECL (84%) showed similarity with proteins in public databases, while for 18 cDNAs (2.4%) no similarity was found. Southern blot assays confirmed the presence of four no match analyzed ORESTES in the genomes of the two strains. No known biological process could be assigned to 39% and 68% of the sequences of ECL and ATVL contigs, respectively. In Proteolysis and Peptidolysis processes 11% and 0.3% of ECL and ATVL ORESTES were allocated, respectively. Additional putative functional differences were observed. The differential abundance of transcripts of some ORESTES was analyzed by northern blot assays in the developmental stages of the strains. Southern blot of the contig ATVL95 originated a hybridization pattern with multiple bands, characteristic of repetitive sequences. This contig corresponds to the transcript of the 195 bp satellite DNA (195 SAT), a repetitive sequence that accounts for 10% of the T. cruzi genome and whose transcription is controversial in the literature. The transcription of the 195 SAT was evidenced by northern blot and RT-PCR experiments. Transcripts of 195 SAT were detected in polyA+ and poly- RNA fractions. These transcripts apparently do not contain the SL sequence, present in trypanosomatid mRNAs. By using oligonucleotides complementary to the two strands of 195 SAT, we concluded that both strands are transcribed. Although it is clear that 195 SAT is transcribed, its biological function remains unknown.
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Yingdong, Zhu. "Analysis of Novel 5'-UTR Polyadenylation Sites in Arabidopsis thaliana." Miami University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=miami1480717686996059.
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Raney, Alexa. "Mechanism of translational regulation of S-adenosylmethionine decarboxylase mRNA by polyamines and an upstream open reading frame /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/9198.
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Jones, Nicola Katherine. "Reading the frame/framing the reader : the negotiation of narrative authority in medieval Italian short story collections." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612253.
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Jennings, Olivia. "Framing a settler literature : a reading of the work of Janet Frame at the postmodern/postcolonial intersection." Thesis, Royal Holloway, University of London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406307.
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Dammann, Kristin [Verfasser], and Ulrike [Akademischer Betreuer] Beisiegel. "Open Reading Frame 2 des LINE 1 Retrotransposons : funktionelle Analyse in Mammaliazellen / Kristin Dammann. Betreuer: Ulrike Beisiegel." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2011. http://d-nb.info/102041720X/34.
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Rippe, Richard Allen. "The molecular cloning and expression of the BPV-2 L-2 open reading frame in Escherichia coli." Diss., The University of Arizona, 1988. http://hdl.handle.net/10150/184402.
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The bovine papilloma virus type 2 (BPV-2) L2 open reading frame (ORF) was cloned into a λ pL promoter expression vector. This clone was shown to express a fusion protein which comprised 75% of the BPV-2 ORF linked to the first 13 N-terminal amino acids of the λ cIl gene product. Antisera was generated against this fusion protein and subsequently used to identify the L2 gene product as a 64,000 dalton protein in BPV-2 virions. It was also demonstrated that the L2 viral protein was present in full caps ids, but only in very limited amounts in empty caps ids. Densitometer analysis indicated that the L2 protein comprised only 8% of the total L1 + L2 "Coomassie blue stainable" protein in full capsids. The antisera was also used to demonstrate that the BPV-2 L2 gene product is antigenically related to the BPV-1 L2 gene product. Finally, an attempt was made to determine the location of the L2 gene product within the capsid structure. Hemagglutination inhibition and enzyme-llnked-immunosorbent- assay data both indicate that the L2 protein is exposed on the surface of the capsid. Immune electron microscopy data was inconclusive in determining the location of the L2 gene product.
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Collins, Nathaniel D. "Studies of pX open reading frame I of Human T-Lymphotropic Virus Type 1 using infectious molecular clones /." The Ohio State University, 1999. http://rave.ohiolink.edu/etdc/view?acc_num=osu1488186329503804.
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Blowers, Tonya. "Locating the self : re-reading autobiography as theory and practice, with particular reference to the writings of Janet Frame." Thesis, University of Warwick, 1998. http://wrap.warwick.ac.uk/36297/.
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The thesis is a three-part study of the theory and practice of autobiography. The writing of the New Zealand novelist, poet and autobiographer Janet Frame (1924-) is used as casestudy throughout, juxtaposed to canonical texts of autobiography (typically written by white western males) which have been used to draw conclusions about the self. Frame's 'autobiographical' writings (in particular her three-volume autobiography, To the Is-Land, An Angel at My Table and The Envoy From Mirror City; and her novels Faces in the Water and Owls Do Cry) are used to suggest a new approach to interpreting both the self in society and the relationship between narrated self and context. Part One is a re-reading of three classic texts of the genre, St.Augustine's Confessions, Dante's Vita Nuova and John Bunyan's Grace Abounding. The assumption that such texts describe an 'autonomous, unitary' male protagonist is thoroughly questioned and the texts are read to reveal instead the characteristics of fragmentation and alterity usually reserved for descriptons of the self in women's autobiographies. The point is emphasised that the narrated self of autobiography must always be precisely located in time and space. In Part Two, the definition of autobiography as genre is explored. Two schools of thought are identified: one which focuses on the contract between reader and writer (Lejeune), the other which highlights that the self is constructed in and through the narrative which purports to represent it (Bruss, Barthes). Frame's writing is then used to test the application of such models. The relationship between 'history' and 'fiction' is discussed as the pivotal distinction on which the notion of autobiography hinges. Through a reading of Frame's autobiographies and Paul Ricoeur's Time and Narrative, the notion of a 'textual contract' as a new definition of autobiography as genre is developed: this definition maintains both the importance of the life outside the text but also the representative nature of narrative to transform that reality within the text. Part Three puts into practice the theory of 'locating the self'. Frame's autobiographies are first analysed through a series of categories of 'belonging': gender, class, race, nationality and coloniality. It is suggested, using Elspeth Probyn's notion of Outside Belonging, that Frame invents and performs the categories of both poet and schizophrenic in order to find a place to belong. Finally, Frame's narrated self is analysed in the very specific context of the local and national writing culture, demonstrating that the narrated self of autobiography is, to a large extent, instructed in society and rehearsed by the author long before she puts pen to paper. The thesis concludes with the notion of autobiography as metaphor which is seen as resolving many of the theoretical dilemmas posed throughout.
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Alderete, John Paul. "Translational effects of mutations and polymorphisms in a repressive upstream open reading frame of the human cytomegalovirus UL4 gene /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/11499.
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Yadav, Kush Kumar. "Genotype 1 hepatitis E virus (HEV) ORF4 protein enhances genotype 3 HEV replication." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574781581580768.
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Chen, Augustine, and n/a. "Translational control mechanisms used by the human Hepatitis B virus : an upstream open reading frame modulates expression of the pregenomic RNA." University of Otago. Department of Biochemistry, 2007. http://adt.otago.ac.nz./public/adt-NZDU20080130.123000.
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The human hepatitis B virus (HBV) is a small hepatotropic virus, which affects approximately 350 million chronic sufferers worldwide. It has a compact 3.2 kbp dsDNA genome encoding four major overlapping genes namely core, polymerase, surface and X required for its replication. The virus synthesises a pregenomic RNA (pgRNA) which functions both as an RNA intermediate for reverse transcription into the DNA genome and as the mRNA for the translation of the core (C) and polymerase (P) proteins. The core overlaps the polymerase gene and is translated at a 10 to 1 ratio. The polymerase gene translated from the P AUG codon is preceded by at least 4 upstream AUG codons (uAUGs), namely C AUG, C1 AUG, J AUG and C2 AUG. Various mechanisms have been implicated in the synthesis of the polymerase protein. This led to the currently accepted model which involves leaky scanning and a reinitiation mechanism in polymerase synthesis.
However, multiple sequence alignment of the pgRNA revealed a short upstream open reading frame (uORF) highly conserved at the nucleotide level in all HBV subtypes and mammalian hepadnaviruses. This previously unreported uORF, designated as C0 ORF in this study is also conserved in its position and length. Past studies have either omitted this uORF in their test constructs or ignored its potential role. The C0 ORF has a conserved weak initiation context and is located within the epsilon structure within the 5' leader of the pgRNA, required for viral encapsidation. Importantly, the C0 ORF precedes and overlaps the core ORF, which may suggest an alternative model in which the core and polymerase may be translated and coordinately regulated.
Fusion of the C0 ORF to luciferase showed for the first time that this uORF is translated through the detection of reporter activity (~20% of C) and also visualisation of the fusion protein via western analysis using anti-C0 and anti-luciferase antibodies. Subsequent removal of the C0 ORF implicated a role in repressing downstream core fusion protein synthesis in HepG2 cells. A similar repression was observed on J expression.
To study the effect of C0 on downstream polymerase translation, a pgRNA-like DNA construct was made and subsequent mutations introduced. Mutation of the C0 AUG led to an increase in initiation at the downstream P AUG. Alteration of the existing weak initiation context to an optimal context which favours stronger initiation consistently showed a potential role for C0 ORF in facilitating reinitiation at certain downstream initiation codons including P AUG. Mutations of other uAUGs preceding the P AUG were also done to better understand their roles in regulating polymerase synthesis. The removal of the C AUG markedly increased expression from the P AUG. This study revealed other internal uAUGs in-frame to the C AUG, namely the C1 and C2 AUGs are also effectively translated, further reducing availability of translating ribosomes to downstream P AUG. Indeed the removal of the C1 and C2 AUGs led to a corresponding increase in initiation from the P AUG. Initiation at the internal J AUG was also reported and its removal showed a significant decrease in expression from the P AUG, consistent with the previous model implicating reinitiation at the P initiation site after translation of the short J ORF. The inhibitory role of the 5 uAUGs prior to the P AUG were confirmed when all were removed, giving rise to translation almost equal to that at C AUG.
Taken together, these results suggest a new model in which the HBV C0 ORF plays a key role in controlling core and polymerase synthesis by repressing core translation and making available more ribosomes to downstream AUGs possibly facilitating translation reinitiation. In addition, the translation of the C0 ORF across the [epsilon] region may also preclude encapsidation, potentially acting as a switch discriminating the pgRNA template between encapsidation and translation. Therefore, the highly conserved [epsilon] region and C0 ORF present an excellent target for molecular based antiviral drugs (antisense oligonucleotides, aptamers, ribozymes) potentially providing new anti HBV drugs.
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Pudupakam, Raghavendra Sumanth Kumar. "Understanding the Role of the Hypervariable Region in the Open Reading Frame 1 of the Hepatitis E virus in Viral Replication." Diss., Virginia Tech, 2011. http://hdl.handle.net/10919/77083.
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Hepatitis E virus (HEV) is a major cause of enterically transmitted acute viral hepatitis in developing countries that lack proper hygienic infrastructure. Hepatitis E is globally distributed and has emerged as an important public health disease in both developing and industrialized countries. HEV is a non-enveloped virus carrying a single-stranded positive-sense RNA genome of approximately 7.200 bp in length. The life cycle of HEV is poorly understood due to the lack of an efficient cell culture system. Animal model systems, including non-human primates, swine, and chickens are being used to study some fundamental aspects of the HEV biology. Recently, novel animal strains of rat and rabbit HEV have been discovered, and whose usage as animal model systems needs to be established. HEV infections in pigs and chickens provide excellent model systems to study the replication and pathogenesis aspects of HEV. Recently, we identified a hypervariable region (HVR) in the open reading frame 1 (ORF1) of HEV. The objectives of this dissertation were to utilize chicken and swine model systems to study the role of HVR in HEV infection in vivo, to determine the effects of HVR on replication of HEV in vitro, and to analyze the effect of exchange of HVR among different genotypes on the replication-competency and virion production in vitro.
Extensive sequence variability in the HVR among HEV strains of different genotypes prompted us to study the dispensability of this region. Initially we constructed two partial deletion mutants of genotype 1 human HEV, hHVRd1 and hHVRd2, with in-frame deletion of amino acids (aa) 711 to 777 and 747 to 761 in the HVR of a sub-genomic GFP HEV replicon. Expression of enhanced green fluorescent protein by the mutant hHVRd2 confirmed the dispensability of amino acid residues 747-761 of the HVR. To confirm our in vitro results, specific-pathogen-free (SPF) chickens were intra-hepatically inoculated with capped RNA transcripts from three avian HEV HVR-deletion mutants: mutants aHVRd1 (Δ557-585), aHVRd2 (Δ612-641), and aHVRd3 (Δ557-641). Chickens intra-hepatically inoculated with the mutants, aHVRd1 and aHVRd2, developed active viral infection as evidenced by seroconversion, viremia, and fecal virus shedding. Mutant aHVRd3, with a larger HVR deletion, was apparently attenuated in chickens. Additionally, we used the swine model system to further verify our results from the chicken study. The infectivity of four genotype 3 swine HEV HVR-deletion mutants, sHVRd1 (Δ712-790), sHVRd2 (Δ722-781), sHVRd3 (Δ735-765), and sHVRd4 (Δ712-765) constructed using the genotype 3 swine HEV as the backbone was determined in SPF pigs. Pigs intra-hepatically inoculated with capped RNA transcripts from the mutants sHVRd2, sHVRd3, and sHVRd4 developed active viral infection, whereas mutant sHVRd1 (Δ712-790), with a nearly complete HVR deletion, exhibited an attenuation phenotype. The data from these studies indicate that deletions in HVR do not abolish HEV infectivity in vitro or in vivo, although evidence for attenuation was observed for HEV mutants with a larger or nearly complete HVR deletion.
To further elucidate the role of HVR in HEV replication, we investigated the effects of serial amino acid deletions in HVR on the replication of HEV. We first constructed a genotype 1 human HEV luciferase replicon by replacing the ORF2 gene that encodes for the capsid protein with the fire fly luciferase reporter gene. Using the backbone of human HEV genotype 1 luciferase replicon, we constructed a series of HVR-deletion mutants with deletions of variable lengths in the HVR. Amino acid deletions Δ711-725, 711-740 and Δ711-750 were engineered at the N-terminus, deletions Δ729-754, Δ721-766, and Δ716-771 were engineered in the central region, and deletions Δ761-775, Δ746-775, and Δ736-775 were engineered at C-terminus of the HVR. The effects of these serial deletions on HEV RNA replication in the human liver carcinoma cell line, Huh7, were examined. Replication levels of mutants carrying these deletions were compared with that of the wild-type HEV in Huh7 cells. We observed that deletions in the HVR did not abolish viral RNA synthesis but substantially reduced the replication levels of viral RNA, as measured by the reporter luciferase activity. To further verify the effects of HVR deletions on viral RNA replication as observed with the genotype 1 human HEV replicon, we subsequently used a genetically-distinct strain of HEV, avian HEV, and constructed an avian HEV sub-genomic luciferase replicon by substituting the ORF2 gene of avian HEV with the fire fly luciferase gene. Avian HEV HVR-deletion mutants Δ557-603, Δ566-595, and Δ573-587 were then engineered using the backbone of avian HEV luciferase replicon. The replication efficiency of the three deletion mutants of avian HEV in chicken liver hepatoma cell line, LMH, was evaluated. Compared with the wild-type avian HEV, the viral RNA synthesis of the avian HEV HVR-deletion mutants was considerably reduced by the HVR deletions. To analyze the impact of the complete HVR deletion on avian HEV infectivity, we constructed an avian HEV mutant with a deletion of the entire HVR region (aaΔ557-603) using the avian HEV infectious cDNA clone as the backbone. After confirming the viability of the complete HVR-deletion mutant in LMH cells, SPF chickens were intrahepatically inoculated with capped RNA transcripts generated from the mutant. None of the chickens inoculated with the complete HVR-deletion mutant showed evidence of HEV infection, indicating that drastic reduction in replication levels due to complete HVR deletion has resulted in the loss of virus infectivity. The results indicated that HVR may have critical residues that may interact with viral/and or host factors and modulate the replication efficiency of HEV.
In the final part of the dissertation research, we sought to determine if the variable sequences of HVR are genotype-specific for in vitro virus replication. By using the genotype 1 human HEV as the backbone, we swapped the HVR of genotype 1 human HEV with the HVRs of the genotype 3 swine HEV and the distantly-related avian HEV to construct two inter-genotypic chimeras, pSKHEV2-Sw and pSKHEV2-Av. Similarly, by using the genotype 3 swine HEV as the backbone, the HVR of genotype 3 swine HEV was swapped with the HVR of genotype 1 human HEV to construct the chimera, pSHEV3-Hu. The viability of these chimeras was tested in Huh7 cells that are permissive for HEV replication. Immunofluorescence assay (IFA) with anti-HEV antibodies revealed that all the three chimeras were replication-competent in Huh7 cells. The infectivity of these chimeras was subsequently evaluated in HepG2 cells. The results showed that exchange of the HVR between different genotypes of mammalian HEVs does not abolish the replication competency and infectivity of HEV. This finding suggests that HVR is not genotype-specific with respect to viral replication and infectivity. The absence of detectable viral antigen in HepG2 cells infected with chimera pSKHEV2-Av suggested a functional incompatibility of the HVR of avian HEV in the mammalian HEV genome.
In summary, we identified a highly variable sequence, HVR, in the ORF1 of the HEV genome, and the sequences of the HVR vary significantly among HEV strains of different genotypes. We found that the HVR contain sequences that are dispensable for virus infectivity both in vitro and in vivo. Deletion analysis of HVR revealed that the region may play a role in modulating the replication efficiency of HEV RNA by interacting with viral and/or host factors. Finally, we demonstrated that HVR is not genotype-specific for virus replication and the region can be functionally replaced between mammalian HEV genotypes for virus replication and virion production in vitro. The results from this dissertation research have important implications for better understanding the biology and mechanism of HEV replication and may aid in our efforts to eventually develop a modified live-attenuated vaccine against HEV.Ph. D.
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Peng, Bee-Zen [Verfasser], Marina [Akademischer Betreuer] Rodnina, Holger [Gutachter] Stark, and Ralf [Gutachter] Ficner. "The Role of EF-G in Translational Reading Frame Maintenance on the Ribosome / Bee-Zen Peng ; Gutachter: Holger Stark, Ralf Ficner ; Betreuer: Marina Rodnina." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2019. http://d-nb.info/119464600X/34.
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Sörensen, Susanne. "In Splendid Isolation : A Deconstructive Close-Reading of a Passage in Janet Frame's "The Lagoon"." Thesis, Högskolan i Halmstad, Sektionen för humaniora (HUM), 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:hh:diva-6090.
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In reading the literary criticism on Janet Frame's work it soon turns out that Frame was deconstructive before the concept was even invented. Thus, deconstruction is used in this essay to close-read a passage in the title story of her collection of short stories, The Lagoon (1951). The main hierarchical dichotomy of the passage is found to be the one between "the sea" and "the lagoon," in which the sea is proven to hold supremacy. "The sea" is read as an image of the great sea of English literary/cultural reference whereas "the lagoon" is read as an image of the vulnerably interdependent, peripheral pool of it, in the form of New Zealand literary/cultural reference. Through this symbolic and post-colonial reading the hierarchical dichotomy between "the sea" and "the lagoon" is deconstrued and reversed. In the conclusion, a post-colonial trace of Maori influence displaces the oppositional relation between "the sea" and "the lagoon."
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Wethmar, Klaus. "C/EBPbeta deltauORF mice - a genetic model for uORF-mediated translational control in mammals." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16314.
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Evolutionär konservierte, kleine offene Leserahmen (upstream open reading frames, uORFs) sind translational aktive Kontrollelemente, die bevorzugt in Boten-Ribonukleinsäuren von Schlüsselgenen zur Regulation von Zellwachstum, Proliferation und Differenzierung vorkommen. In dieser Arbeit wurden Mäuse analysiert, die defizient für das uORF Initiationscodon des Transkriptionsfaktors CCAAT/enhancer binding protein beta (C/EBPbeta-Delta-uORF) sind. Proteinanalysen verschiedener Gewebe zeigten, dass C/EBPbeta-Delta-uORF Mäuse im Gegensatz zu Wildtyptieren nicht in der Lage sind, die kurze, auto-antagonistische C/EBPbeta LIP Isoform zu induzieren. Die verminderte LIP Expression verursachte eine gestörte Differenzierung knochenabbauender Osteoklasten und ging mit einer Zunahme von mineralisiertem Knochengewebe in C/EBPbeta-Delta-uORF Mäusen einher. Nach partieller Hepatektomie führte der Verlust der uORF-vermittelten Induktion von LIP in regenerierenden C/EBPbeta-Delta-uORF Lebern zu einer Überaktivierung C/EBPbeta-regulierter Akute Phase Gene. Im Vergleich zum Wildtyp wiesen Hepatozyten von C/EBPbeta-Delta-uORF Tieren einen verzögerten und abgeschwächten Wiedereintritt in die S-Phase des Zellzyklus auf. Genomweite Genexpressionsanalysen zeigten, dass die verminderte S-Phase Aktivität in regenerierenden C/EBPbeta-Delta-uORF Lebern mit einer persistierenden Repression von Zellzyklusgenen korrelierte, wobei insbesondere die verminderte Expression zahlreicher E2F-regulierter Gene auffällig wurde. Chromatinimmunpräzipitations- und Reportergenexperimente führten zur Entwicklung eines mechanistischen Modells, das eine isoformspezifische C/EBPbeta-Koregulation E2F-kontrollierter Zellzyklusgene vorschlägt. Die Analyse der C/EBPbeta-Delta-uORF Mäuse belegt erstmals die Funktionalität der uORF-gesteuerten translationalen Kontrolle im Säugetier und weist auf eine entscheidende Bedeutung dieses Kontrollmechanismus bei zahlreichen physiologischen und pathopysiologischen Prozessen hin.Evolutionary conserved small upstream open reading frames (uORFs) are translational control elements predominantly prevalent in the 5'' mRNA regions of key regulatory genes of growth, proliferation, and differentiation. This thesis comprises the evaluation of mice deficient for the uORF initiation codon of the transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta-Delta-uORF). Protein analysis of various tissues demonstrated that C/EBPbeta-Delta-uORF mice, in contrast to wildtype control animals (C/EBPbeta-WT), fail to induce translation of the truncated, auto-antagonistic C/EBPbeta LIP isoform. The reduced expression of LIP was associated with impaired differentiation of bone resorbing osteclasts and resulted in an increased bone volume of C/EBPbeta-Delta-uORF mice. After partial hepatectomy the loss of uORF-mediated LIP induction resulted in super activation of acute phase response genes in regenerating livers. Furthermore, C/EBPbeta-Delta-uORF hepatocytes showed a delayed and blunted re-entry into the cell cycle after partial hepatectomy as compared to C/EBPbeta-WT animals. Genome-wide transcript expression analyses revealed that the reduced S-phase activity in regenerating C/EBPbeta-Delta-uORF livers correlated with a persistent repression of cell cycle regulatory genes and showed a remarkable underrepresentation of genes regulated by the E2F family of transcription factors. Chromatinimmunoprecipitations and luciferase reporter gene assays allowed the development of a mechanistic model that suggests C/EBPbeta isoform-specific co-regulation of E2F-controled cell cycle genes. The analysis of C/EBPbeta-Delta-uORF mice validates the functionality of uORF-mediated translational control in vertebrates and suggests a comprehensive role of uORF regulation in physiology and the etiology of disease.
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Mitchell, Andrew. "Discovering Bioactive Peptides and Characterizing the Molecular Pathways that Control Their Activity." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10482.
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Bioactive peptides constitute a major class of signaling molecules in animals and have been shown to play a role in diverse physiological processes, including hypertension, appetite and sleep. As a result, knowing the identity of these molecules and understanding the mechanisms by which they are regulated has basic and medical significance. In this dissertation, I describe the development and application of novel methods for discovering bioactive peptides and the molecular pathways that control their activity. Recent analyses of mammalian RNAs have revealed the translation of numerous short open reading frames (sORFs). However, it is unknown whether these translation events produce stable polypeptide products that persist in the cell at functionally relevant concentrations. In Chapter 1, I describe a study in which we used a novel mass spectrometry-based strategy to directly detect sORF-encoded polypeptides (SEPs) in human cells. This analysis identified 115 novel SEPs, which is the largest number of mammalian SEPs discovered in a single study by more than a factor of 25. We observed widespread translation of SEPs from non-canonical RNA contexts, including polycistronic mRNAs and sORFs defined by non-AUG start codons. We also found that SEPs possess properties characteristic of functional proteins, such as stable expression, high cellular copy numbers, post-translational modifications, sub-cellular localization, the ability to participate in specific protein-protein interactions and the ability to influence gene expression. Taken together, these findings provide the strongest evidence to date that coding sORFs constitute a significant human gene class. In chapter 3, I describe a study in which we combine quantitative in vivo peptidomics, classical biochemical experiments and pharmacological studies in animal models to elucidate the metabolism of the neuropeptide substance P in the spinal cord. We identified two physiological substance P metabolites: the N- terminal fragments SP(1-9) and SP(1-7). Focusing our efforts on the SP(1-9)- producing pathway, we determined that an activity sensitive to the inhibitor GM6001 is the dominant SP(1-9)-generating activity in the spinal cord. We also show that GM6001 treatment causes a nearly three-fold increase in endogenous substance P levels in the spinal cords of mice, highlighting the functional relevance of the pathway blocked by this inhibitor.
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Lee, Yun-Young. "Translational regulation of growth arrest and DNA damage-inducible gene GADD34 via its 5' untranslated region upstream open reading frame during eukaryotic initiation factor 2 alpha phosphorylation." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/17442.
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Endoplasmic reticulum (ER) stress activates an integrated stress response which causes inhibition of overall protein synthesis via phosphorylation of the eukaryotic initiation factor 2alpha (eIF2alpha). However, ER stress also results in selective translation of mRNAs, one of which is a transcription factor ATF4. ATF4 activates transcription of downstream stress-induced genes such as growth arrest and DNA-damage inducible gene 34 (GADD34) under ER stress. The function of GADD34 is to dephosphorylate eIF2alpha by interacting with protein phosphatase 1, thus leading to recovery of overall protein synthesis and translation of stress-induced transcripts through a negative feedback mechanism. In this thesis, we showed that GADD34 is not only transcriptionally induced, but also translationally regulated for maximal expression under ER stress. Translational regulation of GADD34 was mediated by its 5’ untranslated region (5’ UTR), which was found to contain two upstream open reading frames (uORFs) in human and mouse. It was revealed that the downstream uORF2 is required for basal repression and translational upregulation under ER stress, while the upstream uORF1 is dispensable in this regulation. In addition, the uORF2 is readily recognized and translated, but the uORF1 is bypassed by the scanning ribosomes. Further mutational analysis on the GADD34 5’ UTR demonstrated that the uORF2 and the intercistronic region between the uORF2 and the main ORF are sufficient to direct translation when eIF2alpha is phosphorylated. In this process, the amino acid/nucleotide identity of the uORF2 was not required, but its conserved size was important. The sequence conservation within the intercistronic region also was identified, but changing the length and pyrimidine:purine ratio in this region did not significantly affect translational regulation. Finally, we set up in vitro translation systems where cap-dependent translation is compromised by inhibiting ternary complex and eIF4F formation in order to test GADD34 translational regulation. The results from the current thesis suggest that GADD34 translation is mediated through its 5’ UTR via a unique mechanism, which may serve as a model to understand translational regulation of other uORFs-containing mRNAs under cellular stress.
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Peletto, S. "TSE GENETICS IN GOATS. LOOKING OUTSIDE THE PRNP OPEN READING FRAME: MOLECULAR CHARACTERIZATION OF THE PRNP REGULATORY REGIONS AND ASSESSMENT OF THE ROLE OF THE SPRN GENE." Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/169566.
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Susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy of small ruminants, is strongly influenced by polymorphisms of the prion protein gene (PRNP) and breeding programs to increase scrapie resistance in sheep populations have been implemented. A similar approach is not applied in goats yet but it would be desirable. The knowledge of genetic factors, outside the PRNP gene, involved in the modulation of the response to scrapie in goats, could aid to overcome some limitations of future selection programs based solely on PRNP genetics. The main aim of this research project was therefore to look for TSE genetic factors outside the goat PRNP open reading frame by the molecular characterization of the PRNP gene regulatory regions and the assessment of the role of the Shadow of the Prion Protein gene (SPRN), a recently discovered member of the prion family.
In the PRNP regulatory regions, 29 novel polymorphisms were identified within a 3 Kb fragment encompassing the PRNP transcription start site in goats belonging to nine goat breeds coming from Italy, Greece and Spain. Haplotype analysis, performed on the study goats using bioinformatics, predicted 13 haplotypes based on ten polymorphic sites. Most of the polymorphisms with an influence on the presence/absence of transcription factor binding sites were found in intron 1. Functional studies have also demonstrated that a conserved region in the PRNP intron I is able to bind different transcription factors, among which the binding of ELK1 has been assessed by EMSA experiments. Furthermore, the first association study investigating the role of the goat SPRN gene in scrapie occurrence was carried out in the frame of the project. The SPRN genes of goats from several scrapie outbreaks were analysed in order to detect SPRN polymorphisms and to look for association between SPRN alleles/genotypes and the occurrence of scrapie by a case-control study. A significant association with susceptibility for classical scrapie was found for a polymorphism causing the insertion of five nucleotides (602_606insCTCCC) in the 3’UTR. Bioinformatics analyses suggested the hypothesis that the identified indel may contribute to modulation of susceptibility to the disease via gene expression regulation by a miRNA-mediated post-transcriptional mechanism. In conclusion, the results presented in this Doctoral thesis provide novel knowledge on genetic factors outside the caprine PRNP open reading frame investigating their influence in the modulation of scrapie susceptibility. These data could offer a significant contribution to the control of classical scrapie in goats by future breeding programs as well as further research opportunities on other aspects related to the function and pathogenicity of the prion protein.
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Midenius, My, and Kristin Bengtsson. "Förskolebibliotek : Hur förskollärare skapar och använder litterära miljöer i förskolan." Thesis, Linnéuniversitetet, Institutionen för utbildningsvetenskap (UV), 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-69679.
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Syftet med studien är att synliggöra några förskollärares erfarenheter av att skapa och använda litterära miljöer i förskolan för att främja barns intresse för läsning och barnlitteratur. Studien utgår från begreppet förskolebibliotek, samt hur förskolorna arbetar för att skapa miljöer med fokus på läsintresse. Litterära miljöer är ett annat centralt begrepp som beskriver hur miljön samverkar med flera faktorer gällande läsning, detta för att skapa en förståelse kring hur flera faktorer samspelar i en lärmiljö. Frågeställningarna berör hur förskollärare utformar, planerar och genomför aktiviteter i förskolebibliotek med avseende på material samt tillgänglighet, men även hur barnen på egen hand och tillsammans med andra ska utforska den litterära miljön. Det är en kvalitativ studie med semistrukturerade intervjuer som metod, där fem biblioteksansvariga förskollärare från olika förskolor har deltagit. Forskningen i studien riktar sig till den litterära miljöns betydelse för barnen i verksamheten med fokus på hur material, barns inflytande och miljöns utformning samspelar på olika sätt. Under rubriken förskollärare, barn och miljö i samspel lyfts förskollärares förhållningssätt och kompetenser samt barnets motivation till att vilja lära. Forskningen tar även upp läsning och lärande som mer allmänt berör vad läsningen har för betydelse för barns framtida lärande. Resultatet visar att förskollärarna anser att den litterära miljön är viktig och påverkar barns läsintresse, genom att den ger möjlighet till att låta barnen utforska på egna villkor. Ramfaktorerna är en aspekt som tydligt visar sig påverkar arbetet med förskolebiblioteken, då dessa praktiska förutsättningar har inflytande över den dagliga verksamheten på förskolorna.
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Müller, Marcel Alexander [Verfasser]. "Studies on human pathogenic coronaviruses survey for coronaviruses in African bat species and characterization of the novel human coronavirus NL63 (HCoV-NL63) open reading frame 3 (ORF3) / Marcel Alexander Müller." Berlin : Freie Universität Berlin, 2008. http://d-nb.info/1022641670/34.
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Kimbung, Stanley Mbandi. "A computational framework for transcriptome assembly and annotation in non-model organisms: the case of venturia inaequalis." Thesis, University of the Western Cape, 2014. http://hdl.handle.net/11394/4022.
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Philosophiae Doctor - PhDIn this dissertation three computational approaches are presented that enable optimization of reference-free transcriptome reconstruction. The first addresses the selection of bona fide reconstructed transcribed fragments (transfrags) from de novo transcriptome assemblies and annotation with a multiple domain co-occurrence framework. We showed that selected transfrags are functionally relevant and represented over 94% of the information derived from annotation by transference. The second approach relates to quality score based RNA-seq sub-sampling and the description of a novel sequence similarity-derived metric for quality assessment of de novo transcriptome assemblies. A detail systematic analysis of the side effects induced by quality score based trimming and or filtering on artefact removal and transcriptome quality is describe. Aggressive trimming produced incomplete reconstructed and missing transfrags. This approach was applied in generating an optimal transcriptome assembly for a South African isolate of V. inaequalis. The third approach deals with the computational partitioning of transfrags assembled from RNA-Seq of mixed host and pathogen reads. We used this strategy to correct a publicly available transcriptome assembly for V. inaequalis (Indian isolate). We binned 50% of the latter to Apple transfrags and identified putative immunity transcript models. Comparative transcriptomic analysis between fungi transfrags from the Indian and South African isolates reveal effectors or transcripts that may be expressed in planta upon morphogenic differentiation. These studies have successfully identified V. inaequalis specific transfrags that can facilitate gene discovery. The unique access to an in-house draft genome assembly allowed us to provide preliminary description of genes that are implicated in pathogenesis. Gene prediction with bona fide transfrags produced 11,692 protein-coding genes. We identified two hydrophobin-like genes and six accessory genes of the melanin biosynthetic pathway that are implicated in the invasive action of the appressorium. The cazyome reveals an impressive repertoire of carbohydrate degrading enzymes and carbohydrate-binding modules amongst which are six polysaccharide lyases, and the largest number of carbohydrate esterases (twenty-eight) known in any fungus sequenced to date
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Minao, Maya. "Reading Nabokov's framed landscape." 京都大学 (Kyoto University), 2008. http://hdl.handle.net/2433/135504.
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Zhang, Yuanyuan. "TRANSLATIONAL REGULATORY MECHANISMS OF THE RAT AND HUMAN MULTIDRUG RESISTANCE PROTEIN 2." UKnowledge, 2008. http://uknowledge.uky.edu/gradschool_diss/649.
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Multidrug resistance protein 2 (MRP2) is the second member the C subfamily in the superfamily of adenosine triphosphate (ATP)-binding cassette (ABC) efflux transporters. MRP2 is a critical player for generation of bile acidindependent bile flow and biliary excretion of glutathione, glucuronate and sulfate conjugates of endo- and xenobiotics. Dysfunctional expression of MRP2 is associated with Dubin-Johnson Syndrome.
Pathological and physiological states or xenobiotics change the MRP2 expression level. Under some conditions, expression of the human MRP2 and rat Mrp2 proteins are regulated at the translation level. There are several transcription initiation sites in MRP2/Mrp2 gene. The 5’ untranslated regions (5’UTRs) of MRP2/Mrp2 contains multiple translation start codons. The focus of this study, therefore, was investigation of the translational regulatory mechanisms mediated by the upstream open reading frames (uORF) of MRP2/Mrp2.
Using in vitro translation assays and transient cotransfection assays in HepG2 cells, we showed that the rat uORF1 starting at position -109 (relative to the ATG of Mrp2) and the human uORF2 starting at position -105 (relative to the ATG of MRP2) are two major cis-acting inhibitors of translation among the rat and human multiple uORFs, respectively. Translational regulation mediated by the uORFs in the rat Mrp2 mRNA is a combined effect of the leaky scanning model and the reinitiation model, and also results from interaction of the multiple uORFs. In addition, by Ribonuclease Protection Assays (RPA), we detected multiple transcription initiation sites of MRP2/Mrp2 gene in tissues. We also found that the relative abundance of the rat Mrp2 mRNA isoforms with different 5’UTRs differed in the rat liver, kidney, jejunum, ileum, placenta, and lung. This is the first study on the translational regulatory mechanisms of the MRP2/Mrp2 gene.
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Hodgson, Theresa Susan. "Open reading frames 3a and 3b of Infectious bronchitis virus." Thesis, University of Bristol, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422552.
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Shukla, Aditi [Verfasser]. "Analysis of overlapping reading frames in viral genomes / Aditi Shukla." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2016. http://d-nb.info/1103156020/34.
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Ahasan, Mohammad Mainul. "Characterisation of open reading frames m29 and m29.1 in murine cytomegalovirus." Thesis, University of Birmingham, 2008. http://etheses.bham.ac.uk//id/eprint/60/.
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Murine cytomegalovirus (MCMV) in its natural host, the mouse, is an excellent model for studying the biology of cytomegalovirus infection. Mostly this model has been used to study gene homologues of human cytomegalovirus (HCMV). Of the predicted 170 MCMV open reading frames (ORFs) only 78 have significant amino acid identity with genes in HCMV. To better understand the biological mechanisms underlying the differences between the viruses, for example their species specificity and immune evasion genes, MCMV unique ORFs need to be examined. Here the role of m29 and m29.1 ORFs in the MCMV (Smith strain), which have no homology with ORFs of any other cytomegalovirus, have been examined. The m29 and m29.1 ORFs are overlapping and encoded on opposite strands of the double-stranded DNA genome. Sequence analysis over this region showed a discrepancy to the published sequence. An additional G (guanine) nucleotide was found at nucleotide position 36,198 that alters the predicted ORFs, m29 being 242 amino acids shorter and m29.1 210 amino acids longer than the predicted sequence. This was confirmed by sequencing the MCMV Birmingham K181 strain, the Birmingham Smith strain and MCMV wild type isolates- N1, K17A and G4. Transcripts from the newly identified m29 and m29.1 ORFs were confirmed by reverse transcriptase PCR (RT-PCR). They were produced at early (3h) and immediate-early (2h) times post-infection respectively as determined by cycloheximide and phosphonoacetic acid treatment but were continuously expressed up to at least 24h post-infection. 5' and 3'-RACE (rapid amplification of cDNA ends) analysis from m29.1 ORF confirmed the production of a ~2.4 kb transcript and a low abundance spliced transcript from which a 123bp intron had been removed. Mutants of ORF m29 and m29.1 have been produced in which ET recombination was used to introduce stop codon mutations within these overlapping ORFs. This was achieved by single base alterations near to the 5` end of each ORF that prevented translation but not transcription of each ORF individually. Linear dsDNAs containing the mutations were introduced into the Smith MCMV BAC replacing an antibiotic cassette that had been inserted into the gene of interest. Mutant viruses, Rc29 and Rc29.1 respectively, were recovered from these mutant BACs by in vitro passage in tissue culture cells. Revertant virus (Rv29.1) was made by a further 2 step process in which the mutant m29.1 ORF was first replaced by the antibiotic cassette and then by the wt ORF. These mutants were characterized both in tissue culture and in immunocompetent BALB/c and immunodeficient SCID mice. Both mutants produced their expected transcripts but Rc29.1 virus produced no corresponding protein as examined by western blot using an antibody produced in rabbits to bacterially expressed protein. Failure to express the m29 ORF in bacteria and failure of a synthetic peptide to generate rabbit antibodies that bound to denatured m29 protein meant that protein expression of the m29 gene in either mutant could not be determined. Mutant virus Rc29 replicated similarly to wild type virus both in tissue culture and in BALB/c mice. Mutant virus Rc29.1 replicated poorly with lower yields, a delay of about 2-3 days in reaching peak titres and an earlier decline compared to wt and revertant (Rv29.1) virus in tissue culture. Rc29.1 virus also showed delayed replication in the salivary glands of BALB/c mice compared to wt and Rv29.1 viruses and in SCID mice peak titres occurred later and mice became sick and had to be humanely killed approximately 8 days later that mice infected with wt virus. These results suggest that m29 and m29.1 ORFs are dispensable for viral replication in vitro in NIH 3T3 cells and in animal hosts. However, the m29.1 ORF is required for optimal viral growth in vitro and in vivo.