Don Roth - US grants

The long-term goal of this project is to determine the influence of nucleotide binding and protein phosphorylation on the regulation of pathogenesis and define specific mechanisms mediating RNA replication. A host encoded, autophosphorylating, dsRNA dependent protein kinase (p68) has been identified whose activity is regulated in host plants by virus or viroid infection. The protein has biochemical and immunological similarities to proteins implicated in the replication of poliovirus and in the regulation of protein synthesis. Specific aims of this proposal are directed at determining the role of p68 in infected and healthy tissues and at studying the mechanisms by which its activity is regulated. In order to define the function and regulation of this protein, it is being purified and biochemically characterized to determine the gene sequence. Antiserum to the protein is being raised and will be used in localization studies, screening of cDNA libraries and to probe the activity of the protein. Further studies on the purified protein will include characterization of the nucleotide binding and phosphorylation stoichometries, partial sequencing, phosphoamino acid analysis and tryptic peptide mapping in the presence and absence of dsRNA and use of various nucleic acid species to define the mechanism by which dsRNA regulates the activity of p68. If time permits the gene and mRNA sequences will be obtained, yielding information on the total amino acid sequence of the protein and allowing further characterization of regulatory events at the genetic level. Viruses and viroids effectively interact with and regulate host proteins during replication and pathogenesis. The identification and characterization of host proteins directly involved in pathogenesis is important to determine fundamental mechanisms regulating host-pathogen interactions. These studies will provide a foundation to determine how pathogens regulate host proteins, what effect on disease development the interaction has, and provide insight into RNA regulatory mechanisms.***//

Plant viruses and viroids are minimal pathogens and depend upon interaction with key host components during pathogenesis. The temporal and spatial regulation off host factors during initial host-pathogen interactions is conditioned by host recognition of viral/viroid signals and the subsequent transduction of the signal leading to metabolic alterations. The processes by which these agents manipulate host plant proteins are unknown. Protein phosphorylation-dephosphorylation cascades are important post- translational processes which serve to rapidly amplify and transduce specific developmental and environmental signals and coordinate metabolic responses. This laboratory has characterized a plant-encoded protein kinase (p68) whose autophosphorylation is induced in viral and viroid infected plants and in noninfected tissue extracts supplemented with viral dsRNA and artificial dsRNA. The response is signal dependent and occurs in the initial phases of pathogenesis. Studies to date have emphasized biochemical and immunological characterization of p68. In order to better understand the role of p68 it is necessary to begin analysis at the gene level. The investigator has chosen to use both virus- and viroid-host models. Each has distinct attributes. This project will further characterize the significance of p68 activity in host- pathogen interactions and in healthy plants by isolating, sequencing and studying the cDNA and genomic clones of p68. The functional significance also may be indicated by determining the phosphorylation of p68 in tissues challenged with viroid mutants which vary in both RNA stability and pathogenicity. The role of p68 may also be indicated by expression of sense and antisense constructs in transgenic plants. p68 appears to be the only plant protein thus far described that is associated with dsRNA dependent protein kinase activity. The viral/viroid-plant systems are excellent models to define the significance of phosphorylation in host-pathogen interactions as well as to study the role of phosphorylation in healthy plants and how that function is altered during infection. %%% Addition or removal of phosphate groups (phosphorylation, dephosphorylation) from specific proteins plays an important role in the recognition and amplification of environmental and biological signals in all organisms. This process may also have a central role in the way plants perceive and respond to signals generated during attack by pathogens or wounding. The goal of this research is to determine the role of protein phosphorylation in virus-plant interactions. It will focus on a specific protein, "p68", that catalyzes the addition of phosphate to itself in the presence of double stranded RNA from plant viruses. The role of p68 in normal plant physiology and during viral infection will be studied. The processes by which viruses manipulate normal proteins after they infect plants are largely unknown. The results of this research should be an important contribution to basic understanding of how plants respond to viruses.

This request is for funds to purchase a Molecular Dynamics PhosphorImager for use in research and teaching in molecular biology. This instrument will be critical for research applications of at least eight faculty as well as in training undergraduate and graduate students in molecular biology. Autoradiography using storage phosphor technology offers several advantages over traditional detection methods. Quantitation of amounts of radioactive protein or DNA on gels or filters has traditionally been done using X- ray film and densitometry or using scintillation counters. Storage phosphors are up to 100 times more sensitive than X-ray film, enabling measurements well below levels of detection for film. Also, screens are quantitatively accurate over five orders of magnitude as compared to X-ray film which has a linear range over only two orders of magnitude. The new technology is also environmentally advantageous because it eliminates the heavy metal waste disposal problems associated with film processing, and when it replaces scintillation counting it reduces mixed organic and radiochemical waste disposal problems. This technology will enable new experimental approaches, improve quantitation, enhance productivity, and it will afford our students state of the art training.

9513400 Roth Protein phosphorylation-dephosphorylation events condition the regulation of numerous cellular processes in eukaryotes. However, there are few examples in plant systems that provide a model to dissect the functional significance of a phosphorylation-mediated signal transduction pathway. The long term goal of this research is to characterize the role of the plant encoded double-standed (ds) RNA-dependent protein kinase, pPKR, in uninfected and in virus and viroid infected cells. The primary goals of this renewal are: to extend characterization studies of pPKR and to determine its functional significance with respect to regulation of protein synthesis initiation in tobacco and potato plants. The pPKR is part of a regulatory pathway that may be involved in regulation of protein synthesis, cell division and differentiation, and defense against viral infection. Therefore, it is important to understand the role of this protein in plants.

DESCRIPTION (provided by applicant): Rural areas face unique challenges with respect to community understanding of the nature, complexity, and outcomes of basic and clinical biomedical research. This has important consequences in political and social decision-making and establishing learning environments that inspire student understanding of biomedical sciences and selection of careers in the health arenas. These areas are characterized by peer and K-12 school isolation, few appropriate community models including biomedical industries, research institutes and a population lacking an educational background in these areas. In order to overcome these obstacles, the overarching goals of this project are to deliver awareness units to targeted communities, develop and deliver in cooperation with 7, 8th grade science teachers motivational and experiential learning modules with biomedical themes, bolster continuing education for teachers and enhance the graduate enterprise at the University of Wyoming. Multimedia, interactive awareness units will be presented to communities and 7-12 grade assemblies. These will engage the community and contribute to understanding of basic and clinical aspects of biomedical scholarship, its tremendous contributions to society and the need for sustainable support. The learning modules, complete with detailed lesson plans, will prepare students for self-directed learning and will nurture an appreciation for discovery. They will be developed by 7, 8th grade science teachers, graduate fellows and university faculty. The modules will focus on the interdisciplinary nature and unifying concepts of basic and clinical aspects of selected biomedical areas, complement current curricula and align with state standards. Program participants will have opportunities to participate in ongoing workshops, the University Science Fair and summer research apprenticeships. In order to intervene in the prevalent socio-educational culture that now directs students into non-college curricular tracts in the 9th grade, the program will target 7th and 8th grade science classes. Initially, school districts with significant Native American and Hispanic populations have been targeted; however, our long-term goal is to reach all counties in Wyoming, in part, via distance delivery technologies and continuing education workshops. Benchmarks for progress will be established and continually assessed in order to track projected outcomes. Modifications of the methodology will be based upon analysis of quantitative assessment data.

This proposal describes a Track 1 project developed by the University of Wyoming in partnership with several schools with high Native American and Hispanic communities.
The goals of this project are to motivate and prepare students for self-directed learning and to nurture an appreciation for discovery that will lead to careers in science. The project seeks to engage communities to deepen understanding of contemporary STEM issues. The project will focus on the interdisciplinary nature, unifying concepts and ethical considerations of science from the perspective of "What is Alive?" In order to intervene in the prevalent socio-educational culture that now directs students into non-college curricular tracts in the 9th grade, the program will target 7th and 8th grade classes. Initially, communities with significant Native American and Hispanic communities will been targeted; however, the long-term goal will be to reach all school districts in Wyoming, in part, via distance delivery technologies and continuing education workshops. The broad program goals will be attained by meeting four specific objectives:
* Enhance awareness and understanding of contemporary STEM issues, opportunities and ethics by offering motivational units to students and communities.
* Promote STEM knowledge and skills by delivering interdisciplinary curricular units that align with state standards.
* Advance graduate student learning and outcomes.
* Increase sustainable STEM expertise of 7th and 8th grade science teachers.

Knowledge of the nature and relevance of scientific discovery will provoke sustainable student learning, achievement and career choice.

Abstract

Energy and water are major constraints to global prosperity and directly pertinent to global climate change. Their interface is of particular relevance to Wyoming as the leading US state in the exportation of energy but having major water limitations. As a model for illustrating principles of complexity and uncertainty in science, the energy-water interface presents a relevant framework to enhance STEM learning outcomes. In addition, like many rural, frontier states, Wyoming faces distinct challenges with respect to STEM awareness, education, and career opportunities. And a significant proportion of the state population can be categorized as at-risk relative to socio-economic status and educational achievement. These challenges will be addressed through a partnership between schools, the University, and the private sector using a diverse portfolio of activities designed to increase learning outcomes of doctoral fellows, 7-10 grade students and teachers, to inform career choice through motivating STEM experiences, and to institutionalize GK12 goals in the graduate education infrastructure of UW. Specific deliverables from this project include: inquiry based curricular units, summer research experiences, diverse workshops, multiple training events, sustained cyberinfrastructure interactions, and global research opportunities. This program will promote the competitive success of the future STEM workforce by providing training in leadership, communication and project management skills for graduate fellows, by enhancing the educational pipeline through motivational awareness units, through sustained professional development of teachers, and by development of new interdisciplinary graduate programs focused on GK12 objectives.