Thesis (Ph.D.)--University of Wyoming, 2009. / Title from PDF title page (viewed on Apr. 16, 2010). Includes bibliographical references (p. 200-225).

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Philip W. Tucker. Vita. Includes bibliographical references. Also available from UMI.

Thesis (doctoral)--Lund University, 1996. / Added t.p. with thesis statement inserted.

Thèse (Ph. D.)--Université Laval, 2007. / Bibliogr.: f. 208-212. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (Ph.D.)--Memorial University of Newfoundland, 2004. / Bibliography: leaves 201-225.

Thesis (doctoral)--Lund University, 1996. / Added t.p. with thesis statement inserted.

Thesis (Ph. D.)--University of Oregon, 2006. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 80-86). Also available for download via the World Wide Web; free to University of Oregon users.

Murine cytomegalovirus (MCMV) is used as a biological model for human cytomegalovirus (HCMV). Latency, persistence and reactivation are same of the important aspects of the murine model that share analogies with human CMV infections. In order to elucidate the molecular mechanisms leading to these events, in-depth analyses of the murine model are required at the transcriptional level. During the MCMV replication cycle, there is a sequential expression of different regions of the viral genome, hence the transcripts are divided into three kinetic classes; the immediate early (IE), early (E) and late (L). This study presents the analyses of MCMV (Smith strain) transcripts of the major IE and E transcriptional units, and a more detail analysis of one of the major E regions, E10. The IE and E transcripts were studied by probing them with Ctoitplementary DNAs (cDNAs). The cDNAs were prepared from mRNA isolated from the IE and E phases of the viral replication cycle and cloned into the bacteriophage Lambda gt10. Ten E cDNAs were mapped to specific locations of the virus genome, and these represented transcripts from the major E regions in Hindlll fragments A, B, E, F, and I-J. Five E cDNAs, each representing a different major E region, and two IE cDNAs representing the major IE region, were applied as probes in one of the studies to determine the relative transcript levels during the course of infection of 3T3L1 fibroblast cells with MCMV. The major E transcriptional units were investigated further in a study where Northern blots of RNAs, isolated from different phases of the viral replication cycle, were probed with the five E cDNAs. This study revealed transcripts that were temporally regulated since they were present only during the E and usually L phases of the viral replication cycle. In addition, the quantities of these transcripts varied depending on the phase. However, all five cDNAs detected more than one transcript which indicates complex splicing events, overlapping genes, multiple initiation sites and/or the presence of gene(s) in the complementary DNA strand. One of the E cDNAs, E10, corresponding to a transcript from a major E region of Hindlll fragment I-J, was selected for further analysis. The E10 cDNA detected four transcripts of 9.5, 6.9, 4.7 and 2.1 kb in size, which were found to be transcribed from the same DNA strand. The DNA sequence of this E10 cDNA was determined and shown to contain 3223 nucleotides, however it lacked a polyadenylation signal and a poly A tract at the 3' end. The missing 3' terminus, designated as E10-A, was isolated using the polymerase chain reaction (PCJR) method and its DNA sequence of 1422 nucleotides was also determined. The combined sequence of E10 and E10-A (total of 4606 nucleotides) was designated as E10-C and is presented in this thesis. The E10-C cDNA (4.6kbp) most likely represents the 4.7 kb transcript. The E10-C cDNA sequence has one minor and one major open reading frame (ORF). The minor ORF is initiated by the first ATG triplet (nucleotide position 114) while the major ORF is initiated by the second triplet (nucleotide position 155). Since the sequence preceeding the second ATG triplet is in "good context" with regard to the translation initiation consensus sequence, it is most likely that the major ORF is translated. The major ORF (3600 bases) encodes a 1200 amino acid polypeptide, the putative E10 protein of approximately 135 kd in size. A protein close to that size was detected in one of the experiments in which RNAs, that were hybrid-selected by the E10 cDNA and eluted, were translated in vitro. The putative E10 protein lacks homology with any other protein in the data banks (SWISSPRT and GENPEPT). Portions of the viral genomic fragments Hindlll I and J were also sequenced to reveal the orientation of the gene coding for the E10 cDNA and its related transcripts. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Rhodobacter capsulatus photosynthesis gene was isolated by creating in-frame fusions in a lacZ transcriptional/translational vector, and selecting for those that directed oxygen-regulated levels of β-galactosidase in R. capsulatus. One lacZ fusion isolate was used to identify an open reading frame (ORF) of unknown function and flanking sequences that promoted initiation of transcription. Interposon mutagenesis experiments identified the ORF as the bchC gene, which encodes an enzyme that catalyses the penultimate step in the biosynthesis of bacteriochlorophyll a, and also showed that the bchC gene formed an operon with the bchA gene. The nucleotide sequence of this bchC gene and its 5' regulatory region were determined. The deduced amino acid sequence showed that the bchC gene encodes a 33 kDA protein that has hydrophobic segments that could interact with a lipid membrane, and that this putative BchC protein contains a potential bacteriochlorophyll a binding site. Deletion analysis, S1-nuclease protection, and primer extension experiments showed that promoter activity was associated with sequences to which a 5' end mapped, and that these sequences had significant similarity to the proposed promoter regions of several other R. capsulatus photosynthesis genes. RNA blotting and S1-nuclease protection end-mapping experiments using bipartite probes provided direct evidence that the mRNA transcripts of the bchCA operon overlap those of the two flanking operons, the crtEF and the puf operons, such that the crtEF, bchCA, and puf operons may be cotranscribable, and that RNA polymerase may initiate transcription at one of several promoters. The significance of these overlapping mRNAs was evaluated using two interposon mutant strains, one that prevented crtEF transcripts from overlapping those the bchCA and puf operons, and the other that prevented both crtEF and bchCA transcripts from overlapping those of the puf operon. The results suggested that transcriptional readthrough stimulates promoter activity. Moreover, a pufB::lac'Z fusion could be expressed from the bchCA promoter equally as well as from the puf promoter, suggesting that these overlapping transcripts are functionally significant in the chromosomal context. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

All sequenced archaeal genomes encode a general transcription factor, TFE, which is highly conserved and homologous to the alpha subunit of the eukaryotic transcription factor TFIIE. TFE functions to increase promoter opening efficiency during transcription initiation, although the mechanism for this is unclear. The N-terminus of TFE contains a common DNA binding motif, a winged helix. At the tip of this winged helix is a highly conserved region of aromatic amino acids that is close to DNA during initiation. TFE activation can compensate for mutations in another transcription factor, TFB2, which is homologous to TFIIB. P. furiosus encodes two paralogs of the eukaryotic RNA polymerase II transcription factor TFIIB: TFB1 and TFB2. TFB2 lacks a portion of the highly conserved N-terminus, and functions in transcription complexes at a lower efficiency than TFB1. It has been demonstrated that the presence of TFE is able to assist in transcription with TFB2 in vitro bringing its efficiency to almost TFB1 levels. Thus, TFB2 provides a unique opportunity to evaluate the function of the TFE winged helix in transcription. In this study the aromatic patch of the TFE winged helix was mutated to test its role in activation of TFB1 and TFB2-containing transcription complexes, because this aromatic patch is required for full TFE activity especially when NTP concentrations are low.