1985 — 1988 |
Lohr, Dennis E |
K04Activity Code Description: Undocumented code - click on the grant title for more information. |
Combined Structural/Functional Analysis of Gene Control @ Arizona State University-Tempe Campus
The potential functional significance of chromatin structural features associated with the (putative) control regions of three yeast genes, galactokinase, histone H2B and 35S rDNA will be evaluated, in order to determine whether there is a causative relationship between these structural features and the (unknown) features involved in transcriptional aspects of the control of gene expression in eukaryotic organisms. In parallel, work will be directed towards elucidating the physical forces involed in stabilizing anomalous control chromatin features and towards determining if there are any anomalous DNA conformational flexibility features associated with control sequence DNA. In vitro mutagenesis studies will be used to assess the dependence of control chromatin features and control sequence DNA conformational flexibility on DNA sequence. Various assays of gene expression level (viability, mRNA production, normal regulation) will be correlated with the physical data. A new technique for precise assessment of in nucleo DNA-protein interactions will be developed, to increase the sensitivity level for detecting anomalous chromatin structural features. A hybrid gene system will be used to try to develop a technique to obtain functional and structural data on one of the genes in a highly repeated gene set (35S rDNA). The goal of all these experiments is to gain some understanding of how eukaryotic cells control gene expression. An understanding of the bases for the control of gene expression is absolutely essential in understanding and solving a number of outstanding health problems, for example, cancer, aging and processes like abnormal development, which affect humans and other multicellular organisms. However, the latter systems are very complex. If one can gain an understanding of some of the bases for control of gene expression in a system like yeast, which has fewer complexities and is amenable to very favorable manipulations, this knowledge will be useful in helping to suggest approaches which can lead to understanding in the more complex systems.
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0.958 |
1987 — 1990 |
Lohr, Dennis E |
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. |
Structural Analysis of Ribosomal Chromatin @ Arizona State University-Tempe Campus
The experiments in this proposal will characterize the chromatin structure of the genes and promoter elements of the 5S and 35S ribosomal genes in yeast. Nuclease and chemical cleavage will characterize the primary level of chromatin structure across the approximately 5 kb region which includes the genes and their promotors, by indirect end label analysis, applying both low resolution (nucleosome patterns and locations, hypersensitive regions) and high resolution (nuclear footprinting of DNA/protein interactions) approaches. A technique allowing similar analysis in vivo will be developed and applied. Several interesting regions including the defined promoters and terminators for the ribosomal genes will be of particular interest. The role of aspects associated with the higher orders of chromatin structure (matrix attachment, supercoiling) in the function of these elements will also be assessed. The structural data will be used as an assay to isolate trans-acting factors involved in the proper function of the gene control elements for these genes. We will utilize a unique system which allows one to probe a single riboxomal gene in the midst of the approximately 100 other copies in the yeast cell. By determining the unambiguous structure of these elements in various expression states, we should learn more about how these elements function in gene control. The combined structural/functional analysis of this well defined system will give novel insights on the relation of chromatin structure to control of gene expression in eukaryotes. An understanding of the bases for the control of gene expression is absolutely essential in understanding and solving a number of outstanding health problems, for example, cancer, aging and processes like abnormal development, which affect humans and other multicellular organisms. However, the latter systems are very complex. If one can gain an understanding of some of the bases for control of gene expression in a system like yeast, which has fewer complexities and is amenable to very favorable manipulations, this knowledge will be useful in helping to suggest approaches which can lead to understanding in the more complex systems.
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0.958 |
2003 — 2006 |
Lohr, Dennis Taguchi, Aileen Woodbury, Neal [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Characterization of Dna/Protein Interactions At the Single Molecule Level @ Arizona State University
The goal of this work is to explore, at the single molecule level, three different types of DNA/protein interactions: 1) Binding interactions between proteins and DNA with a particular emphasis on the role of one-dimensional diffusion, 2) Generation of loops in DNA by protein complexes that bind at two or more nonadjacent sites, 3) Protein interactions which disrupt base stacking by pulling a base out of the stacking configuration with neighboring bases. Each of these processes will be explored at both the level of population distributions (relative populations of different geometries or conformations of the DNA/protein complex at equilibrium) and in terms of dynamics (times required for interconversion between conformations or geometries, again primarily at equilibrium). Single molecule fluorescence spectroscopy is well suited for such measurements. The primary emphasis will be placed on three DNA/Protein systems. These are the interaction of intercalating dyes with DNA as a model system for protein/DNA interaction and the study of various DNA binding domains with DNA with an emphasis on searching for binding sites and protein/DNA interactions which result in DNA looping. Some work will be performed on a final and ambitious project, monitoring the action of DNA polymerase at the single molecule level. This work will be done in collaboration with Dr. Reha-Krantz at the University of Alberta, who will provide some of the DNA templates and constructs required for the experiments. The project will be closely connected to an existing IGERT program at the university, and undergraduate students will be heavily involved in the research.
This project is jointly funded by the Physics and Chemistry Divisions in the Directorate for Mathematical and Physical Sciences and the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.
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1 |
2006 — 2007 |
Thorpe, Michael (co-PI) [⬀] Lohr, Dennis Woodbury, Neal [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
"Sger: Merging Single Molecule Spectroscopy and Molecular Simulation". @ Arizona State University
This SGER project is designed to take the first steps in merging the single molecule nucleosome dynamics system developed by the PI Woodbury with a new approach to simulating the dynamics of large molecular systems on the millisecond and longer time scale. In general, single molecule fluorescence spectroscopy suffers from the lack of a solid theoretical approach to backing out fundamental physical parameters from observed dynamics. The nucleosome is a case in point. There are several recent publications from several laboratories showing that measurements of single molecule dynamics can be performed on nucleosomes. However, beyond a qualitative understanding of the range and frequency of motion, little can be concluded. What is needed is a theoretical model to predict stochastic single molecule dynamics based on structural and thermodynamic properties. By comparing experiment and simulation, the work in this project is designed to determine if it is possible to directly test the physical model, refining the key parameters.
This project is jointly funded by the Division of Physics and the Division of Chemistry in the Mathematical and Physical Sciences Directorate and the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.
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