Thesis (Honors)--College of William and Mary, 2009. / Includes bibliographical references (leaves 67-73). Also available via the World Wide Web.

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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

The major histocompatibility complex of the rat (RT1 complex) encodes two sets of class II molecules referred to as RT1 B and RT1 D. The RT1 Bα gene was isolated from a Sprague-Dawley (RT1b) rat genomic library using a rat RT1 Bα chain cDNA as a hybridization probe. The coding and the majority of the intron DNA sequence was determined. The structure of the RT1 Bα gene is equivalent to that of H-2 and HLA a chain genes. Comparison of the nucleotide and predicted amino acid sequences of the RT1 Bα gene to those of the H-2 and HLA genes revealed a high degree of overall sequence conservation. However, two regions of the first external domain (a1), residues 19-23 and 45-78, exhibit marked sequence variation. Two blocks of conserved nucleotide sequence were identified in the 5' promoter region of the RT1 Bα gene that have been described in all MHC class II genes sequenced to date. These conserved sequences may be involved in the co-ordinate regulation of expression of class II genes. The cloned RT1 Bα gene was efficiently transcribed when transfected into mouse L cells. / Medicine, Faculty of / Medical Genetics, 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.

The genome of the hyperthermophile archaeon Pyrococcus furiosus encodes two transcription factor B (TFB) paralogs, one of which (TFB1) was previously characterized in transcription initiation. The second TFB (TFB2) is unusual in that it lacks recognizable homology to the archaeal TFB/eukaryotic TFIIB B-reader (also called the B-finger) motif. TFB2 functions, though poorly, in promoter-dependent transcription initiation. Domain swaps between TFB1 and TFB2 showed that the low activity of TFB2 is determined mainly by its N terminus. The low activity of TFB2 in promoter opening and transcription can be partially relieved by transcription factor E (TFE). The results indicate that the TFB N-terminal region, containing conserved Zn ribbon and B-finger motifs, is important in promoter opening and that TFE can compensate for defects in the N terminus through enhancement of promoter opening. Archaeal RNA polymerase requires two transcription factors for initiation: TBP, which binds to TATA boxes, and TFB, which binds TBP and DNA, recruits RNAP and helps initiate transcription. Archaeal TFBs usually contain a conserved B-reader sequence homologous to the eukaryotic B-reader motif in their N-terminal domains. This region is involved in the assembly of the transcription complex, promoter melting and in transcription start site determination but its position and orientation relative to promoter DNA during initiation is not clear. In this study the positioning of the TFB B-reader relative to DNA was determined by cross-linking using TFB variants substituted with photoactivatable unnatural amino acids. The results demonstrate that the B-reader is in close proximity to the transcription start site on the template but not the non-template strand in transcription initiation complexes. Furthermore, the position of the B-reader varies between closed and open promoter complexes, and between open promoter and early initiation complexes. Thus the archaeal B-reader sequence is poised to interact with promoter DNA in a dynamic fashion, and is likely playing a role in positioning the template-strand in an open pre-initiation complex.

The hyperthermophile archaeon, Pyrococcus furiosus encodes two eukaryotic TFIIB family proteins, TFB1 and TFB2. TFB1 is very similar to TFIIB in terms of sequence homology and function, whereas TFB2 is unusual as it is missing highly conserved sequences in its N-terminal domain that are present in TFIIB and TFB1. Despite this, TFB2 is effective in transcription process, albeit with lower efficiency compared to TFB1. Other archaea also contain multiple TFBs, but unlike Pyrococcus furiosus TFB2, these multiple TFBs have higher sequence homology to each other and have similar transcription efficiencies. Photochemical cross-linking experiments have shown that the B-reader of TFB in archaea and TFIIB in eukaryotes is close to transcription start site and is very important in RNAP recruitment to promoter DNA and transcription start site selection. Thus the lack of the highly conserved B reader region in P. furiosus TFB2 presents the opportunity to further study the functional importance of this region. In this study several amino acids in N-terminal domain of TFB2 were mutated with photoactivable unnatural amino acid p-benzoyl L- phenylalanine (pBpa) and the proximity of TFB2 relative to DNA was determined by photochemical cross-linking experiments. The results showed that TFB2 interacts with DNA near the TATA box via its C-terminal domain, and interacts with both strands of DNA near the transcription start site via its divergent B-reader and the B-linker sequences. The B-reader loop region is close to transcription start site and interacts with the transcribed strand of promoter DNA while the B-linker strand cross-links with the non-transcribed strand. Some of the amino acids in between the B-reader loop and the B-linker strand region in TFB2 are seen to cross-link both the transcribed and the non-transcribed strand. Thus, despite the absence of strong homology to conserved B-reader and B-linker sequences, TFB2 is likely to interact with DNA in the transcription bubble and facilitate in transcription initiation.

Basic helix-loop-helix transcription factors play important roles in pathways that result in the formation of such tissues as skeletal muscle, the nervous system, pancreas and heart. A recently discovered B-cell restricted bHLH transcription factor activated Bcell factor 1 (ABF -1) is suspected to play a role in the regulation of B cell differentiation. ABF -1 was initially characterized as being a transcriptional repressor because it was able to prevent the transactivation abilities of E47 in HeLa cells and the region responsible was hypothesized to reside within the C-terminal portion. This study has further mapped the repression domain to the C-terminal portion bearing the bHLH domain. In addition, we report the discovery of a region in the N-terminus that has a secondary negative effect on the transactivation ability of E2A. To complement these studies, the subcellular localization and nuclear localization signal of GFP-tagged ABF -1 proteins was performed using fluorescent microscopy. These studies suggest that ABF-1 exerts it function through the bHLH domain and the region of the nuclear localization signal lies within amino acids 71-103. Taken together, the ability of the C-terminal end to repress E2A mediated transcription may be answered through the conservation of two amino acids, serine and lysine, located at amino acid positions 124 and 125 in the first helix of the HLH domain. This conservation is seen in known transcriptional repressors, such as ceABF -1, MyoR/musculin and capsulin, but is absent in all other identified bHLH proteins.

The basic helix-loop-helix (bHLH) family oftranscription factors consists of proteins involved in cellular proliferation and differentiation. The HLH structure plays a key role in protein-protein dimerization and with the DNA target sites, referred to as E boxes containing the consensus DNA sequence CANNTG. One class of mammalian class I bHLH proteins includes products of the E2A gene, which result from alternative splicing (E12, E47, and ITF), E2-2 and HEB. E2A proteins have also been detected in most cell lines with high levels of expression in lymphoid- and pancreatic cells. It has also been demonstrated that E2A is required for B cell maturation, T cell development and has been shown to function as tumor suppressors. To date, an E2A-interacting bHLH transcription factor largely restricted to activated B lymphocytes, called ABF -1, was isolated using the two-hybrid system. ABF -1 is the only B cell restricted bHLH protein isolated. ABF-1/E2A heterodimers have been detected in B lymphocytes. In these studies, the mapping of the ABF-1 promoter and the critical 5' regulatory elements that control ABF-1 gene expression were analyzed through 5' deletional analysis. 5' -DNA flanking pieces of the promoter region were created through PCR and inserted into a promoterless cloning vector containing the firefly luciferase reporter gene. RT -PCR analysis and anchored PCR was utilized to demonstrate the transcriptional activity of the - promoter region of the ABF-1 gene. Transient transfections were completed to determine critical regulatory sequences. The promoter location was confirmed through computer analysis of the nucleotide sequence and deletional analysis.