Mountain Scholar

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Colorado State University, Fort Collins 26836
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Open Textbook Library Archive 25
A limited number of titles are available here. To see all OTL titles, please visit the Open Textbook Library at https://open.umn.edu/opentextbooks. Only Open Textbook Library staff have access to all OTL Archive titles held in Mountain Scholar.
University Press of Colorado 1067
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Access status: Open Access ,
Structural and biochemical insights into the role of the nucleosome in transcriptional activation in yeast
(2003) White, Cindy LuAnn Holland, author; Luger, Karolin, advisor; Stargell, Laurie A., committee member; Peersen, Olve, committee member; Laybourn, Paul J., committee member; Anderson, Oren P., committee member
In vivo transcription occurs in the context of highly compacted chromatin. The fundamental repeating element in chromatin and the primary level of DNA compaction is the nucleosome core particle, which comprises 147 base pairs of DNA wrapped around a histone octamer. The extreme compaction of DNA found in the confines of the nucleus has profound implications for our understanding of transcription regulation, since chromatin must allow transcriptional access to specific regions of the DNA molecule while keeping the remaining regions repressed. Approaching these problems by using yeast as a model organism, we determined the three-dimensional structures of the macromolecular complexes in question, and studied nucleosomal dynamics using biochemical and biophysical methods. First, we solved the crystal structure of the nucleosome core particle from the yeast Saccharomyces cerevisiae. Compared to higher eukayotes, the structure of the yeast nucleosome suggests a less stable structure, as well as a mechanism for looser nucleosomal compaction, since yeast nucleosome crystals demonstrate an altered packing within the crystal lattice. Biophysical analysis of the stability of yeast nucleosomes confirmed that they are less stable compared to those of Xenopus laevis. We then investigated the role of poly (dA-dT) DNA sequence elements, found in many yeast promoter regions, on transcriptional activation within a nucleosome context. Our results suggest that these rigid DNA tracts form nucleosomes with the same affinity as a strong positioning DNA sequence, and the formed nucleosome is not destabilized by the presence of the poly (dA-dT) tract. We also find that these elements create areas of greater nucleosomal DNA accessibility, which leads to a more efficient binding of a transcription factor. Finally, we show that a transcription factor can bind directly to nucleosomal DNA near the dyad causing partial dissociation of the DNA ends, but does not bring about the dissociation of the underlying histone octamer. Taken together, these studies shed light on the interactions between transcription factors and nucleosomes, and thus allow us to better analyze the interplay between chromatin packaging and transcriptional regulation at the molecular level.
Access status: Open Access ,
In vivo and in vitro studies of the role of chromatin in PHO5 regulation
(2003) Wongwisansri, Sriwan, author; Laybourn, Paul J., advisor; Bamburg, James R., committee member; Stargell, Laurie A., committee member; Van Orden, Alan, committee member
The Saccharomyces cerevisiae PHO5 gene has served as an excellent model to study transcription in the chromatin environment. Transcription of PHO5 is regulated by phosphate availability through chromatin dynamics. Under activating conditions, positioned nucleosomes on the PHO5 promoter are remodeled by the coordinated function of transcriptional activators Pho2p and Pho4p. This dissertation describes the use of yeast chromatin assembly system to investigate the mechanism of PHO5 regulation. Several transcription-related activities were reconstituted on the chromatin template, including ATP-dependent chromatin remodeling and histone acetylation, which have been demonstrated to be required for activation of numerous genes. Transcriptional activators Pho2p and Pho4p by themselves were capable of binding their recognition sequences in the chromatin-assembled DNA. Chromatin reconfiguration on the PHO5 promoter required Pho2p, Pho4p and the ATP-dependent activity in the nuclear extract. However, chromatin remodeling activity alone was not sufficient to bring about PHO5 transcriptional activation. We showed that the missing activity was histone acetyltransferase. Transcriptional activator binding to chromatin, ATP-dependent chromatin remodeling and histone acetylation were separable events but all were required to activate in vitro transcription of PHO5. In vivo study on the role(s) of histone deacetylase Rpd3p in PHO5 regulation suggested that Rpd3p functions on the PHO5 promoter through controlling acetylation level of the histone tails. Rpd3p also has an indirect role in PHO5 regulation through its effect on phosphate uptake. I have demonstrated that Rpd3p regulates the turn over rate of the phosphate transporter Pho84p, which functions upstream of PHO5 in the PHO pathway. RPD3 deletion resulted in early recycling of Pho84p, resulting in a phosphate uptake defect and accelerated activation of genes involved in phosphate accumulation. Both in vitro and in vivo studies described in this dissertation have contributed significantly to the field of PHO5 regulation. The yeast chromatin reconstitution system has proved to be a very useful tool to study mechanism of transcription regulation. Chromatin reconstitution on bead-bound DNA (described in chapter 5) should provide an additional tool to investigate further details in the mechanism of transcription regulation in a chromatin context.
Access status: Open Access ,
Nucleation and growth studies of polycrystalline covalent materials
(2003) Yun, Jungheum, author; Dandy, David S., advisor; Watson, A. Ted, committee member; Parkinson, Bruce, committee member; Belfiore, Laurence, committee member
The chemical vapor deposition of different covalent polycrystalline materials—including diamond, silicon carbide, and carbon nitride—in stagnation flow reactors was rigorously simulated to determine the nucleation and growth mechanism s of these materials. Kinetic models were used to predict the rates of gas-phase and surface chemistry, the temperature and velocity profiles, potential gaseous film growth precursors, the time evolution of nucleation and intermediate layer formation, and the morphological evolution of continuous polycrystalline films. Numerical studies were also carried out to determine the dependence of the kinetics of nucleation and subsequent polycrystalline film growth on operating conditions. The calculated results for carbon nitride deposition indicate that the experimentally measured bond types in the carbon nitride films must result from chemical bond rearrangement occurring on the deposition surface or in the bulk phase once gaseous film growth precursors, including C, CH2, CH3, C2H2, N, NH, NH2, HCN, and H2CN, are adsorbed. Of these precursors, C and CH3 dominate the carbon contribution to carbon nitride film growth, and atomic nitrogen is the principal nitrogen bearing species. When the evolution rates of a silicon carbide intermediate layer and diamond clusters are calculated by accounting for gas-phase and surface reactions, surface and bulk diffusion, the mechanism for intermediate layer formation, and heterogeneous diamond nucleation kinetics, it is predicted that higher adsorption energies, in the range of 3.7 to 4.5 eV, lead to larger surface adatom densities, lower saturated nucleation densities, and larger silicon carbide intermediate layer thicknesses. The intermediate layer thickness becomes saturated while the growing diamond nuclei still cover a very small fraction of the silicon carbide. Reports of heteroepitaxial diamond nucleation without silicon carbide intermediate layer formation may be readily explained by a significant decrease in the intermediate layer thickness at lower substrate temperatures and at higher diamond nucleation densities. Further, the results of the morphology evolution model reveal that the crystallographic texture and surface morphology—surface roughness, film texture, and grain size—of polycrystalline silicon carbide films, as well as diamond films deposited on the silicon carbide layer, are strongly dependent upon the saturated nucleation density, the deposition condition, and film thickness.
Access status: Open Access ,
Thorstein Veblen and Joseph Schumpeter as precursors of economic sociology
(2003) Wunder, Timothy A., author; Stanfield, J. R., advisor
This work is designed in order to compare the ideas of two of the twentieth century's most influential economists, Thorstein Veblen and Joseph Schumpeter. Veblen and Schumpeter's works are analyzed and compared to each other in order to examine fundamental similarities and differences about important questions upon methodology and Capitalism. The questions, which are asked, are derived from modern explorations into Economic Sociology and are designed to place the works of Veblen and Schumpeter into that region of thought but this study should appeal to anyone interested in twentieth century economic thought. One of this study's major purposes is to fill a gap in the current literature of the history of economic thought. Presently research into the literature shows that there is no extensive comparison of the works of Veblen and Schumpeter and such a comparison would allow a better understanding of the two authors with respect to their beliefs. There are many articles that have been written about either Veblen or Schumpeter that will mention the other author in passing, but there are few articles that have actually attempted to compare the two authors. This study is motivated by the belief that the works of Veblen and Schumpeter still offer many insights to modem social scientists. Many within Economic Sociology are questioning the usefulness of neoclassical analysis and this study will serve to offer a countering method of observing the economy more in line with modem Economic Sociology. Another important purpose of this study is to offer into evidence the similarities that exist in the works of the two authors. The comparison of Veblen and Schumpeter, which is at the heart of this work, would be difficult, if not impossible if such similarities were not present. In this regard it will be shown that the works of Schumpeter diverge dramatically from mainstream theory. His concept of the individual, methodology, and Capitalism clearly place him closer to Veblen than to the economic mainstream. This study offers the close examination of his work that is needed in order to make clear this distinction.
Access status: Open Access ,
Molecular recognition of human CBP by retroviral transcriptional activators
(2003) Vendel, Andrew C., author; Lumb, Kevin, advisor; Kennan, Alan, committee member; Rithner, Chris, committee member; Stargell, Laurie, committee member
HIV-1 Tat is required for the expression of the viral genome. The coactivator and acetyltransferase CREB binding protein (CBP), and the paralog p300, are recruited to the HIV-1 promoter by Tat to aid viral expression. Here we identify the interacting domains of Tat and CBP. Circular dichroism and pulldown assays show that full-length Tat binds to the KIX domain of CBP, but not to the C/H1 or CR2 domains of CBP. Circular dichroism and NMR studies of Tat deletion mutants localize the KlX-binding domain of Tat to the N-terminal 24 residues of Tat. Transient cotransfections demonstrate that exogenous KIX behaves as a dominant negative to Tat-mediated transcription in human T-cells, suggesting that Tat and KIX interact in vivo. These findings indicate that Tat targets the KIX domain of CBP and provide insight into the molecular interactions involved in regulating HIV-1 gene expression. Chemical-shift perturbation mapping with heteronuclear nuclear magnetic resonance spectroscopy was used to identify the surface of human KIX that interacts with Tat. It was found that that Tat binds to the c-Jun/MLL binding surface of KIX, as opposed to the CREB binding site. The results provide new insight into the molecular basis of the assembly of protein complexes involving p300/CBP and Tat during HIV gene expression. The HTLV-1 transcription activator Tax is required for viral replication and pathogenesis. In concert with human CREB, Tax recruits the human transcriptional coactivator and histone acetyltransferase p300/CBP to the HTLV-1 promoter. Here we investigate the structural features of the interaction between Tax and the KIX domain of human p300/CBP. Circular dichroism spectroscopy, nuclear magnetic resonance chemical-shift perturbation mapping and sedimentation equilibrium show that a subdomain of Tax (residues 59-98) binds KIX. Chemical-shift perturbation mapping reveals that the Tax-binding surface of KIX is distinct from that utilized by CREB, and corresponds to the site of KIX that interacts with MLL, c-Jun, and HIV-1 Tat. Sedimentation equilibrium shows that Tax and the phosphorylated KID domain of CREB can simultaneously bind KIX to form a ternary 1:1:1 complex. The results provide a molecular description of the concerted recruitment of p300/CBP via the KIX domain by Tax and phosphorylated CREB during Tax-mediated gene expression.