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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Transformation and Plaque Forming Ability of Adenovirus Type 5 E1A Insertion Mutants

McGrory, Joel 10 1900 (has links)
The E1 region of the group C adenoviruses is able to induce oncogenic transformation of rodent cells in culture and is also necessary for the efficient transcription of other viral genes. The proteins of the E1a transcription unit have been shown to play a pivotal role in both of these activities. In order to better understand the regions of the E1a proteins required for transformation and viral growth a program of random insertion mutagenesis was undertaken by D. S. Bautista to help identify important domains. The 39bp linker insertion oligonucleotides were designed to encode a 13 amino acid in frame insertion in one orientation or a closed reading frame insertion in the opposite orientation. As well the insertion mutants could be collapsed by digestion with BamHI to generate a 2 amino acid in frame insertion. Using this method all three types of mutants were generated at 18 different sites within the E1a coding sequences. The purpose of this project was to assay these E1a mutants for the ability to cooperate with EJb in the transformation of primary baby rat kidney cells using DNA-mediated transfection and also to 'rescue' the mutants into infectious virus and study the ability of the mutant virus to replicate on HeLa cells. Results showed that only closed reading frame mutations upstream of the unique region were completely negative for transformation. Conversely, 13aa or 2aa insertions outside of the unique region impaired but did not abolish transformation. However 8 of the 9 insertions in the unique region of the 289R protein of E1a were defective for transformation of BRK cells in E1a plus E1b DNA-mediated transformation assays. To determine whether the unique region played a direct role in the transformation process or if it had an indirect role such as the transactivation of E1b the transformation assay was carried out using selective media that allow~ growth of foci transformed by E1a alone. Results from this assay showed that the unique region mutants combined with E1b were able to transform with about the same efficiency as E1a alone. The transformation assay was also performed using the unique region mutants in an E1a only background cotransfected with the EJ-ras oncogene. Results from these experiments showed that the unique region mutants in an E1a only background could cooperate with ras in transformation as well as wild-type E1a. From these results it was concluded that the unique region does not play a direct role in transformation by E1 but is required for the efficient expression of E1b which results in wild type transforming frequencies. The actual role of E1b in transformation is unknown. The insertion mutants were also 'rescued' back into infectious virus to study their effect on the ability of the viruses to replicate. The results showed that only viruses in which the unique region was either eliminated or altered were defective for growth on HeLa cells. Transactivation assays carried out by D. Bautista showed results which were comparable to results of infectivity assays. Taken together the results suggest that only the unique region is required for transactivation and only the ability of E1a to transactivate is of importance for viral replication in HeLa cells. / Thesis / Master of Science (MS)
2

Analyzing the Biological Role of Human DREF and its Interaction with the Adenovirus E1A Protein

Radko, Sandi 11 September 2015 (has links)
Early region 1A (E1A) protein is the first protein expressed following viral infection. E1A proteins initiate the cell cycle in infected cell by altering cellular gene expression and also activate expression of other viral genes enabling viral replication. The C-terminus of E1A is the least-characterized region of the protein, with few known binding partners, however DREF has been identified as a novel binding partner. My studies have determined that DREF directly binds to E1A and has a role in the virus life cycle. DREF is a restriction factor for virus growth and is a component of viral replication centres. DREF is SUMOylated and SUMOylation appears to affect localization to viral replication centres. DREF co-localizes with PML bodies and subcellular distribution of DREF is altered by the presence of E1A. This work provides a platform to study the role of DREF in uninfected cells, and in HAdV biology and its possible role in antiviral response. / October 2015
3

p53 activity during adenovirus infection / Die Aktivität des Tumorsuppressors p53 in Adenovirus-infizierten Zellen

Savelyeva, Irina 30 October 2009 (has links)
No description available.
4

Characterisation of CtBP : A Co-Repressor of Transcription that Interacts with the Adenovirus E1A Protein

Sundqvist, Anders January 2001 (has links)
<p>In this study, adenovirus E1A has been used to target and analyse the transcriptional function of the cellular C-terminal Binding Protein (CtBP).</p><p>Transcription is a complex biochemical process that represents a major regulatory step in gene expression. Formation of condensed chromatin by histone deacetylation and inhibition of efficient assembly of the transcription machinery are hallmarks of transcriptional repression. During a virus infection, an extensive modulation of the host cell gene expression in favour of viral gene expression can be observed. For example, the transcription regulatory E1A protein from adenovirus has been proven to be a valuable research-tool in characterising cellular proteins controlling eukaryotic gene expression.</p><p>Expression of a CtBP-binding peptide, encoded by the second exon of E1A, de-repressed transcription from a broad range of promoters, suggesting that CtBP functioned as a repressor of transcription. Artificial promoter recruitment of CtBP, by using different Gal4-fusion proteins, confirmed that CtBP functioned as a repressor. Repression of transcription by Gal4E1A-recruited CtBP was efficiently prevented by a CtBP binding competent E1A peptide, indicating that E1A relieved CtBP mediated repression by displacing CtBP from the promoter. Furthermore, Gal4CtBP repressed both basal and activated transcription in a distance dependent manner, suggesting that CtBP might repress transcription by interfering with the assembly of the basal transcription machinery. Interestingly, CtBP was found to interact with histone deacetylase-1 (HDAC-1) both <i>in vivo</i> and <i>in vitro</i> and endogenous CtBP could also recruit histone deacetylase activity. This might indicate that histone deacetylation was involved in CtBP mediated repression. However, Gal4CtBP mediated repression was insensitive to inhibition of histone deacetylase activity, suggesting an alternative function of HDAC-binding in CtBP mediated repression.</p><p>In conclusion, this work demonstrates that CtBP can act as a general repressor of activated and basal transcription. Furthermore, although CtBP was shown to recruit histone deacetylase activity the relevance of this binding remains unclear.</p>
5

Characterisation of CtBP : A Co-Repressor of Transcription that Interacts with the Adenovirus E1A Protein

Sundqvist, Anders January 2001 (has links)
In this study, adenovirus E1A has been used to target and analyse the transcriptional function of the cellular C-terminal Binding Protein (CtBP). Transcription is a complex biochemical process that represents a major regulatory step in gene expression. Formation of condensed chromatin by histone deacetylation and inhibition of efficient assembly of the transcription machinery are hallmarks of transcriptional repression. During a virus infection, an extensive modulation of the host cell gene expression in favour of viral gene expression can be observed. For example, the transcription regulatory E1A protein from adenovirus has been proven to be a valuable research-tool in characterising cellular proteins controlling eukaryotic gene expression. Expression of a CtBP-binding peptide, encoded by the second exon of E1A, de-repressed transcription from a broad range of promoters, suggesting that CtBP functioned as a repressor of transcription. Artificial promoter recruitment of CtBP, by using different Gal4-fusion proteins, confirmed that CtBP functioned as a repressor. Repression of transcription by Gal4E1A-recruited CtBP was efficiently prevented by a CtBP binding competent E1A peptide, indicating that E1A relieved CtBP mediated repression by displacing CtBP from the promoter. Furthermore, Gal4CtBP repressed both basal and activated transcription in a distance dependent manner, suggesting that CtBP might repress transcription by interfering with the assembly of the basal transcription machinery. Interestingly, CtBP was found to interact with histone deacetylase-1 (HDAC-1) both in vivo and in vitro and endogenous CtBP could also recruit histone deacetylase activity. This might indicate that histone deacetylation was involved in CtBP mediated repression. However, Gal4CtBP mediated repression was insensitive to inhibition of histone deacetylase activity, suggesting an alternative function of HDAC-binding in CtBP mediated repression. In conclusion, this work demonstrates that CtBP can act as a general repressor of activated and basal transcription. Furthermore, although CtBP was shown to recruit histone deacetylase activity the relevance of this binding remains unclear.
6

Inhibition of NFKB by adenovirus E1A in induction of macrophage senstivity [sic] and reduced tumorigencity [sic] in vivo /

Morris, Kristin Renee. January 2006 (has links)
Thesis (Ph.D. in Immunology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 129-141). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
7

Characterisation of CtBP : a co-repressor of transcription that interacts with the adenovirus E1A protein /

Sundqvist, Anders, January 2001 (has links)
Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 3 uppsatser.
8

Modulation of Adenovirus E1A Activities by the Cellular Corepressor CtBP

Johansson, Cecilia January 2006 (has links)
<p>Adenovirus E1A is needed to activate early viral genes and induce cell cycle progression to optimise the conditions for viral replication. This is mostly achieved through interactions between the first exon of E1A and cellular transcriptional regulatory proteins. The carboxy terminus of E1A binds the cellular corepressor of transcription C-terminal Binding Protein (CtBP), resulting in derepression of CtBP target genes. </p><p>Inducible stable U2OS cell lines were established, expressing wild type E1A (E1Awt) and a mutant unable to bind CtBP (E1A∆CID). Low inducible levels and loss of protein expression after prolonged induction together with induction of apoptosis were consistent with the fact that wild type E1A is a cytotoxic protein and correlated with the ability of CtBP to repress proapoptotic genes. E1A∆CID did not induce apoptosis and could be expressed at high levels for prolonged time periods. Moreover, the binding of CtBP contributed to E1A-induced activation of viral E1B and E4 genes, through possible targeting of Sp1 and ATF transcription factors.</p><p>In a micorarray study on mRNA levels in E1A-expressing cells, several genes consistent with the tumour suppressive and apoptotic properties of E1Awt were identified as differentially expressed. Furthermore, the differences between the two cell lines correlated with the presence of binding sites for CtBP-interacting transcription factors in the promoters of regulated genes, enabling the possible identification of new CtBP target genes. </p><p>Finally, a molecular characterisation of the CtBP mechanism of repression revealed that positioning proximal to the basal promoter element was required for efficient repression, suggesting that CtBP interferes with the basal transcriptional machinery. Two separate domains were identified in CtBP, conferring transcriptional repression and activation when expressed alone, achieved through their interaction with HDACs and HATs, respectively. However, together they cooperate to ensure maximal repression through recruitment of histone deacetylase and inhibition of histone acetyl transferase activity.</p><p>Together, these data shows important modulation of E1A activities by the binding of CtBP and suggests the involvement of acetylation/deacetylation complexes for the regulation of E1A function.</p>
9

Modulation of Adenovirus E1A Activities by the Cellular Corepressor CtBP

Johansson, Cecilia January 2006 (has links)
Adenovirus E1A is needed to activate early viral genes and induce cell cycle progression to optimise the conditions for viral replication. This is mostly achieved through interactions between the first exon of E1A and cellular transcriptional regulatory proteins. The carboxy terminus of E1A binds the cellular corepressor of transcription C-terminal Binding Protein (CtBP), resulting in derepression of CtBP target genes. Inducible stable U2OS cell lines were established, expressing wild type E1A (E1Awt) and a mutant unable to bind CtBP (E1A∆CID). Low inducible levels and loss of protein expression after prolonged induction together with induction of apoptosis were consistent with the fact that wild type E1A is a cytotoxic protein and correlated with the ability of CtBP to repress proapoptotic genes. E1A∆CID did not induce apoptosis and could be expressed at high levels for prolonged time periods. Moreover, the binding of CtBP contributed to E1A-induced activation of viral E1B and E4 genes, through possible targeting of Sp1 and ATF transcription factors. In a micorarray study on mRNA levels in E1A-expressing cells, several genes consistent with the tumour suppressive and apoptotic properties of E1Awt were identified as differentially expressed. Furthermore, the differences between the two cell lines correlated with the presence of binding sites for CtBP-interacting transcription factors in the promoters of regulated genes, enabling the possible identification of new CtBP target genes. Finally, a molecular characterisation of the CtBP mechanism of repression revealed that positioning proximal to the basal promoter element was required for efficient repression, suggesting that CtBP interferes with the basal transcriptional machinery. Two separate domains were identified in CtBP, conferring transcriptional repression and activation when expressed alone, achieved through their interaction with HDACs and HATs, respectively. However, together they cooperate to ensure maximal repression through recruitment of histone deacetylase and inhibition of histone acetyl transferase activity. Together, these data shows important modulation of E1A activities by the binding of CtBP and suggests the involvement of acetylation/deacetylation complexes for the regulation of E1A function.
10

Functional Characterization of the Evolutionarily Conserved Adenoviral Proteins L4-22K and L4-33K

Östberg, Sara January 2014 (has links)
Regulation of adenoviral gene expression is a complex process directed by viral proteins controlling a multitude of different activities at distinct phases of the virus life cycle. This thesis discusses adenoviral regulation of transcription and splicing by two proteins expressed at the late phase: L4-22K and L4-33K. These are closely related with a common N-terminus but unique C-terminal domains. The L4-33K protein is an alternative RNA splicing factor inducing L1-IIIa mRNA splicing, while L4-22K is stimulating transcription from the major late promoter (MLP). The L4-33K protein contains a tiny RS-repeat in its unique C-terminal end that is essential for the splicing enhancer function of the protein. Here we demonstrate that the tiny RS-repeat is required for localization of the protein to the nucleus and viral replication centers. Further, we describe an auto-regulatory loop where L4-33K enhances splicing of its own intron. The preliminary characterization of the responsive RNA-element suggests that it differs from the previously defined L4-33K-responsive element activating L1-IIIa mRNA splicing. L4-22K lacks the ability to enhance L1-IIIa splicing in vivo, and here we show that the protein is defective in L1-IIIa or other late pre-mRNA splicing reactions in vitro. Interestingly, we found a novel function for the L4-22K and L4-33K proteins as regulators of E1A alternative splicing. Both proteins selectively upregulated E1A-10S mRNA accumulation in transfection experiments, by a mechanism independent of the tiny RS-repeat. Although L4-22K is reported to be an MLP transcriptional enhancer protein, here we show that L4-22K also functions as a repressor of MLP transcription. This novel activity depends on the integrity of the major late first leader 5’ splice site. The model suggests that at low concentrations L4-22K activates MLP transcription while at high concentrations L4-22K represses transcription. So far, characterizations of the L4-22K and L4-33K proteins have been limited to human adenoviruses 2 or 5 (HAdV-2/5). We expanded our experiments to include HAdV-3, HAdV-4, HAdV-9, HAdV-11 and HAdV-41. The results demonstrated that the transcription- or splicing-enhancing properties of L4-22K and L4-33K, respectively, are evolutionarily conserved and non-overlapping. Thus, the sequence-based conservation is mirrored by the functions, as expected for functionally important proteins.

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