Translational efficiency is dictated in part by the availability of charged transfer RNA. Depletion of aminoacylated tRNAs (e.g. during recombinant protein expression) can increase translational errors and associated stress responses. Here, the role of tRNAs as regulators of gene expression was explored through development of synthetic, tRNA-regulated gene circuits, and through an investigation of the impact of tRNA aminoacylation on endogenous gene expression. Synthetic gene circuits initially explored the use of dominant negative alleles of the release factor eRF1 to modulate stop codon readthrough and translationally regulate gene expression. Mutant eRF1 proteins exhibited only a six-fold stimulatory effect on stop codon readthrough. The dominant negative phenotype was rescued partially by overexpression of eRF1, but not eRF3. Ultimately the severity of growth inhibition by these eRF1 alleles limited their utility in synthetic gene circuit design. A novel synthetic circuit was then implemented that utilised TetR interaction with a TetR-inducing peptide in order to control the expression of a suppressor tRNA, and thus a luciferase reporter gene. Using a parameterised mathematical model, the promoter configuration of the circuit was successfully optimised, allowing suppressor tRNAs to regulate the production of luciferase in both feedforward and positive feedback modes of operation. The effects of charged tRNA levels on the global translation network were dissected by regulating the S.cerevisiae glutamine tRNA synthetase gene GLN4 using a tet-off doxycyclineregulated promoter. tRNA synthetase depletion caused the activation of the Gcn4 amino acid starvation response due to accumulation of uncharged glutamine tRNAs. Doxycycline GLN4 shut-off caused increased amino acid production, and decreased ribosome biosynthesis at the transcriptomic and proteomic level, and further physiological changes proposed to result from compromised translation of glutamine-rich regulatory proteins. tRNA overexpression in the GLN4 depletion strain successfully caused altered competition between different isoacceptor tRNA types for their cognate synthetase resource. Together, these results support a growing understanding of tRNA as a key modulator of translation and gene expression in synthetic and natural systems.

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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

Dysgenic male recombination (MR) induced by the P strains T-007 and OKI rarely, if ever, occurs in the heterochromatin of chromosome two. One possible explanation is that the lack of heterochromatic exchange is due to the highly condensed chromatin in this region. Butyrate (a suspected modifier of chromatin structure) induced significant levels of heterochromatic MR in dysgenic hybrids derived from crosses involving two different P strains. This finding is consistent with the hypothesis that chromatin structure can influence the insertion and excision of P elements and hence MR. Analogous experiments were performed using third chromosome suppressor of variegation (Su(var)) mutations. Neither suppressor mutation induced any heterochromatic MR, suggesting that the mode of action of these Su(var) genes is different from, and more specific than, that of butyrate. One of the mutations (325) which is thought to influence meiotic recombination frequencies, causes some alterations in euchromatic MR in crosses involving the OKI strain. The other mutation, 318, affects neither meiotic nor dysgenic recombination. Su(var) 325 is the first known "factor" to influence meiotic and dysgenic recombination similarly. / Science, Faculty of / Zoology, Department of / Graduate

The purpose of this study was two-fold. The first objective was to determine if Saccharomyces cerevisiae is a useful system for investigating the expression of T-DNA (it takes several months to obtain sufficient bacteria-free transformed plant tissue to investigate T-DNA transcription). A short fragment of T-DNA carrying T-DNA gene 7 was cloned into a yeast plasmid in an attempt to investigate the expression of gene 7 in yeast. The second objective was to determine the significance of a heat shock related sequence identified in the 5¹ region of T-DNA gene 7. Primer extension analysis, SI nuclease mapping, and Northern hybridizations indicate that transcription of T-DNA gene 7 in yeast is different from that of transcription of gene 7 in crown gall tumors. Transcription is different because the distance between the TATA box and the transcription initiation sites must be at least 40 nucleotides in yeast. Therefore, Saccharomyces cerevisiae does not appear to be a useful system for investigating the expression of T-DNA. Crown gall tumors were subjected to a number of stress agents, including heat shock, to determine the significance of the heat shock related sequence identified in gene 7. Primer extension analyses indicate that only cadmium and mercury have a significant effect on the expression of T-DNA gene 7. Although gene 7 responds to cadmium and mercury, the increase in transcription does not appear to be heat shock or metallothionein related, indicating that another mechanism is involved in the enhanced transcription of T-DNA gene 7 in crown gall tumors. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

An Agrobacterium-mediated transformation system of purple raspberry ‘Amethyst’ was established after a series of experiments that determined the effect of genotype, inoculum density, and co-cultivation time on transformation. In this study, a plant regeneration protocol was established for ‘Joan J’ and ‘Polana’ (the regeneration protocol of ‘Amethyst’ was previously developed). Agrobacterium-mediated transformation was conducted for all three cultivars. The minimum killing level of hygromycin B and kanamycin was determined. Inoculum density and co-cultivation time were optimized. Polymerase chain reaction (PCR) verified a successful transformation of ‘Amethyst’ with the frequency of 3.3 ~ 4.4 % when leaves were infected with Agrobacterium EHA105 at the cell density of OD600 0.3 and co-cultivated for 3 days in the medium with 25.0 mg∙l-1 kanamycin. Transgenic lines with the PtFIT gene were hydroponically grown under iron sufficiency or deficiency. The real-time quantitative PCR verified the gene expression in response to iron sufficiency and deficiency conditions.

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.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg, 17 May 1994. / Two black South African subjects presenting with hereditary elliptocytosis were investigated and the defect defined as Spol/74, a previously described spectrin variant leading to defective heterodimer self-association and instability of the erythrocyte membrane. [Abbreviated Abstract. Open document to view full version]