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DC Field | Value | Language |
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dc.contributor.advisor | Capone, John P. | en_US |
dc.contributor.author | Syroid, Edward Daniel | en_US |
dc.date.accessioned | 2014-06-18T16:35:27Z | - |
dc.date.available | 2014-06-18T16:35:27Z | - |
dc.date.created | 2010-06-19 | en_US |
dc.date.issued | 1994-08 | en_US |
dc.identifier.other | opendissertations/1759 | en_US |
dc.identifier.other | 3142 | en_US |
dc.identifier.other | 1363985 | en_US |
dc.identifier.uri | http://hdl.handle.net/11375/6447 | - |
dc.description.abstract | <p>The ability of suppressor (su⁺) tRNAs to abrogate the effects of otherwise-lethal nonsense mutations forms the basis of a genetic screen for the identification of essential genes. The availability of numerous nonsense su⁺ tRNA strains of E. coli and yeast led to the rapid development of prokaryotic and yeast genetics. The recent availability of mammalian nonsense su⁺ tRNA genes has afforded not only the opportunity of applying similar strategies towards the advancement of mammalian viral and cellular genetics, but also the opportunity to examine the regulation of transcription by RNA polymerase III (polIII) in vivo. This thesis comprises two parts. The initial aim of this work involved the development and use of mammalian systems of nonsense mutation suppression to examine the mechanisms of eukaryotic class II gene expression and regulation. Herpes simplex virus type-1 (HSV-1) was chosen as a model system since HSV-1 genes are expressed in a regulated cascade. A natural progression of this work involved the study of the molecular mechanisms of mammalian polIII transcription in vivo and in vitro. Initial work involved the identification and propagation of conditional-lethal nonsense mutants of HSV-1 using a mammalian cell line which can be induced to express high levels of a human amber su⁺ tRNA^(Ser) gene as the permissive host in a simple host-range screening system. Several HSV-1 mutants have been isolated from virus stocks mutagenized with nitrous acid and have been partially characterized. Such mutants exhibit a number of interesting temperature- and/or host-range-dependent phenotypes including variations in growth, cell-to-ceil spread, and plaque morphology. Mutant A7'(A9-2) is particularly interesting since this isolate is defective in an essential function required for HSV-1 late gene expression. Isolate A7'(A9-2) is a potential nonsense mutant since a growth enhancement of 100 fold is exhibited under conditions of suppression. To enhance the utility of mammalian systems of nonsense mutation suppression, a novel and more generally applicable means of efficiently regulating the expression of a su⁺ tRNA gene was developed. Stringent control of su⁺ tRNA gene expression is required since constitutive high levels of nonsense suppression activity is deleterious to mammalian cells. The Escherichia coli (E. coli) lac operator/repressor system has been adapted to confer inducibility upon the human su⁺ tRNA^(Ser) gene both in vivo and in vitro. Lac repressor protein, bound to its cognate lac operator site appropriately positioned upstream of the su⁺ tRNA gene, stringently inhibited tRNA gene expression in vivo, as determined by quantitating suppression of an indicator gene nonsense mutation following transfection of mammalian cells. However, su⁺ tRNA gene expression was quantitatively restored with the allosteric inducer isopropylthio-β-D-galactoside (lPTG). Similarly, lac repressor effected the complete inhibition of lac operator-linked tRNA gene transcription in vitro in HeLa cell nuclear extracts by precluding the assembly of an active polIII transcription complex, and this inhibition was reversible with IPTG. The results demonstrate that a DNA binding protein positioned upstream of a eukaryotic tRNA gene may impart transcriptional regulation upon tRNA gene expression, a novel and particularly intriguing finding in view of the intragenic nature of tRNA gene promoters. The ability to conveniently and reversibly manipulate su⁺ tRNA gene expression may foster rapid progress in the development of mammalian nonsense su⁺ tRNA genetics, perhaps through the establishment of additional mammalian cell lines which conditionally express a variety of nonsense su⁺ tRNA genes. The second aspect of this thesis involved an examination of the molecular mechanisms that govern transcription by polIII which, in mammalian systems, has remained largely refractory to study through conventional approaches. A novel approach was adopted and involved use of the lac repressor protein as a reagent to probe various stages of polIII transcription to define the promoter disposition and functional properties of mammalian polIII transcription complexes. The effect on tRNA gene transcription, of varying the position of lac repressor protein upstream and downstream of the tRNA gene, was examined in vitro in HeLa cell nuclear extracts. Lac repressor differentially and reversibly interfered with different stages of transcription complex assembly and productive initiation. Such analyses have identified distinct functional and structural properties of mammalian polIII transcription complexes, and have provided a view of the spatial arrangement of mammalian polIII preinitiation complexes. The mammalian polIII transcription complex extends at least 35 nucleotides (nts) upstream, and to within 10 nts downstream of the tRNA gene. Moreover, sequences directly upstream of the coding region remain accessible to DNA binding proteins throughout multiple rounds of transcription. The results illustrate a number of potential mechanisms whereby DNA binding factors may modulate transcription initiation by polIII. The lac operator/repressor system has also afforded a novel means of examining transcription elongation and termination by mammalian polIII. Lac repressor protein, appropriately positioned downstream of the tRNA gene coding region, reversibly blocked elongation by polIII in vitro in HeLa cell nuclear extracts, thereby resulting in either the formation of paused polIII ternary transcription complexes, a subset of which maintained the ability to undergo direct transcriptional readthrough of the lac repressor obstacle, or in premature transcription termination. The formation of paused polIII ternary complexes effectively mediated the inhibition of tRNA gene transcriptional activity, which was restored upon removal of the repressor-mediated block with IPTG. The results demonstrate that DNA binding proteins can modulate elongation and termination by polIII in vitro, and suggest that conditional factor-mediated blocks to elongation by polIII may function in vivo to attenuate transcription of class III genes. The ability to selectively arrest elongation by polIII at defined positions within the tRNA gene transcription unit has permitted the identification of discrete functional properties of paused mammalian polIII ternary complexes and may afford a novel strategy for the generation of homogeneous populations of stalled polIII ternary complexes, in which to study their biochemical properties.</p> | en_US |
dc.subject | Biochemistry | en_US |
dc.subject | Biochemistry | en_US |
dc.title | Nonsense Suppressor tRNAs in the Study of Class II and Class III Gene Expression and Regulation | en_US |
dc.type | thesis | en_US |
dc.contributor.department | Biochemistry | en_US |
dc.description.degree | Doctor of Philosophy (PhD) | en_US |
Appears in Collections: | Open Access Dissertations and Theses |
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