1985 — 1986 |
Dahlquist, Frederick Capaldi, Roderick (co-PI) [⬀] Sprague, George (co-PI) [⬀] Sprague, Karen Matthews, Brian (co-PI) [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Acquisition of An Automated Dna Synthesizer @ University of Oregon Eugene |
0.915 |
1985 — 1993 |
Sprague, Karen U |
K04Activity Code Description: Undocumented code - click on the grant title for more information. R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Control of Trna Gene Expression in Bombyx Mori
The aim of the proposed work is to investigate the mechanism of transcription by silkworm RNA polymerase III in vitro. Much of our effort will focus on a novel transcription component, Factor X, that our laboratory has recently isolated. This factor is an essential part of the machinery that transcribes eukaryotic tRNA and 5S genes. It is generally assumed that such machinery is composed entirely of polypeptides. The remarkable feature of Factor X is that it appears to be an RNA rather than a protein. The finding of an RNA with transcription activity is unprecedented. The discovery of Factor X came from our systematic fractionation of the components required for tRNA and 5S RNA transcription in vitro. It is distinct from the components previously resolved by our laboratory and others: polymerase III, and the transcription factors A, B, C, and D. Factor X appears to be a nucleic acid because it is resistant to heat, detergent, phenol, and protease, but sensitive to nuclease. It appears to be an RNA because it is resistant to DNAse, but sensitive to RNAse and alkali. The specific aims of this proposal are: (1) to identify the critical segments of the large tRNA(Ala)C promoter that are essential for function, (2) to purify Factor X, as well as the other silkworm transcription factors in order to establish their identities unambiguously, (3) to assign specific functions to Factor X and the other transcription factors by (a) determining whether they participate in factor-template and factor-factor interactions, (b) determining which phase of the cell cycle requires Factor X, and (c) constructing mutant versions of Factor X to test specific hypotheses for its function.
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1 |
1985 — 1990 |
Sprague, Karen U |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Basis For Selective Expression of Trna Genes
To analyze the molecular mechanisms involved in selective gene expression in differentiated eukaryotic cells, we are studying the alanine tRNA genes from the silkworm, Bombyx mori. These genes are interesting because their products, alanine tRNAs, are accumulated in a tissue-specific fashion. One species of alanine tRNA (tRNACAla) is present in all tissues and is therefore designated constitutive, while the other species (tRNASGAla) is found only in the silkgland. Our goal is to discover the molecular basis for the tissue-specific appearance of alanine tRNA in the silkworm. An investigation into the mechanism responsible for this phenomenon addresses the general problem of gene control during eukaryotic cellular differentiation. We think it likely that tissue-specific regulation of polymerase III transcription is the basis of the differential accumulation of the two alanine tRNAs. We have discovered both cis-acting and trans-acting elements that differentially affect transcription of these templates. Because these effects are large, and because they can be analyzed in vitro, we are in a strong position to determine the precise molecular basis of differential tRNAAla gene expression. We propose a detailed in vitro analysis of the components that interact in trans with the tRNAASG1a gene. In brief, we will define the transcription component(s) that differentially affects transcription of tRNACAla and tRNASGAla genes. We will ask whether the gene-specific component exerts its effect by binding to the gene itself, or by binding to part of the general transcription apparatus, and we have proposed experiments to determine what step in the overall transcription reaction is differentially affected on the two templates. To determine the relevance of our in vitro analysis to the situation in vivo, we will use in vivo footprinting to examine transcription complexes formed in intact silkworm cells. In addition, with antibodies raised against gene-specific transcription components, we will determine the tissue distribution of these factors to see if modulations in their level could account for the activity of tRNASGAla genes in the silkgland.
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1 |
1986 — 1988 |
Sprague, George (co-PI) [⬀] Sprague, Karen Matthews, Brian (co-PI) [⬀] Von Hippel, Peter [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Acquisition of An Ultracentrifuge and a Counter @ University of Oregon Eugene |
0.915 |
1991 — 1999 |
Sprague, Karen U |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanism of Selective Expression of Trna Genes
The goal of this work is to discover the mechanism of differential transcription in vitro of two kinds of tRNA genes. These genes encode alanine tRNA in the silkworm, Bombyx mori, and they differentially regulated in this organism. One class (tRNAAlaC genes) encodes alanine tRNA that is common to all silkworm cell types. The other class (tRNAAlaSG genes) encodes a distinct alanine tRNA that is found only the silkgland. The proposed study addresses the general problem of eukaryotic gene control, and the specific problem of transcription by RNA polymerase III. Since RNA polymerase III acts in all cells of all eukaryotes, understanding its function has broad biological and medical significance. The transcription properties displayed by tRNAAlaSG genes in vitro are very different from those displayed by tRNAAlaC genes -- and probably provide the basis for differential regulation in vivo. Therefore, a mechanistic analysis of these in vitro properties is biologically relevant. The interesting features of tRNAAlaSG transcription in vitro are 1.) It is extremely inefficient (relative to tRNAAlaC transcription) under standard conditions, and 2.) It is as efficient as tRNAAlaC transcription under special conditions. These two states could correspond to the inactivity of tRNAAlaSG genes in most silkworm cell types, and their high level of activity (equivalent to tRNAAlaC genes) in the silkgland. The molecular basis of both of these properties will be investigated. Specifically, the proposed experiments will identify the interaction(s) with the transcription machinery, and the step in the transduction cycle that is less efficient for tRNAAlaSG templates. When the defect in tRNAAlaSG transcription has been localized, the mechanism for overcoming it will be analyzed. To determine whether specific stimulation of tRNAAlaSG transcription in vitro is due to a qualitative or a quantitative change in the transcription machinery, the stimulatory component(s) will be resolved from the remainder of the transcription machinery, and tested for its specific activity on the two kinds of tRNAAla genes.
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1 |
1993 |
Sprague, Karen U |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
1993 Gordon Research Conference On Nucleic Acids @ Gordon Research Conferences
Partial support is requested for the 1993 Gordon Research Conference on Nucleic Acids, to be held on June 14-18 at the New Hampton School, New Hampton, New Hampshire. The purpose of the Conference is to assemble for discussion a diverse group of scientists who actively work at the forefront of research on nucleic acids. There is rapid progress in our understanding of the molecular mechanisms by which nucleic acids participate in complex biological processes. An important aspect of the conference on nucleic acids is that it brings workers in structural and chemical research together with more biologically oriented investigators. As subspecialties have grown over the years, and developed their own meetings, the Nucleic Acids Conference has continued to play a seminal role because it brings together scientists with such a wide range of perspectives and expertise. A broad range of participants will be sought for the 1993 Conference, with scientific accomplishment being the main criterion for admission. In addition to participation within the lecture/discussion format, conferees will be encouraged to participate less formally in poster presentations and in a Molecular Structure Workshop.
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0.915 |