Alan Myers - US grants

The thermodynamic properties of electrolyte mixtures are studied. The isoactive-solvent theory developed in this work is based upon a semi-grant canonical ensemble, for which the solvent activity is the independent variable and the thermodynamic excess functions are expressed in mole ratios. Preliminary evaluations indicate that vapor-liquid equilibria predicted by the theory are in excellent agreement with experiments on ternary systems containing mixed solutes and/or mixed solvents. The theory is being tested for various classes of systems under a wide range of conditions and compared with established theories and correlations. Isopiestic experiments from 25 to 100 C are performed for aqueous solutions containing non-volatile electrolytes. In these measurements, the activity of the solvent is determined simultaneously for single and mixed solutes. Therefore, the theory can be compared directly with experiment without interpolating between experimental points.

A theory encompassing steric exclusion and surface heterogeneity will be formulated for mixed gas adsorption on microporous solids such as zeolites and activated carbon. The theory contains experimentally determined fractional exclusion constants. Besides the theory, three additional studies based on the theory are undertaken: (1) characterization of microporous solids; (2) extension of the theory to adsorption of liquid mixtures; and (3) development of a simple, rapid gravimetric technique for measuring mixture adsorption. Successful conclusion of the study would lead to a major advance in adsorption theory.

The rho genes comprise an evolutionarily conserved family with significant homology to the ras oncogene family. The implied structural similarity of rho and ras proteins suggests that the two families evolved from a common ancestral gene, and that the proteins share common biochemical functions. In the yeast Saccharomyces cerevisiae, similar mutations in either rho or ras genes prevent the normal sporulation response to nutrient limitation. These and other observations suggest that rho and ras proteins function in signal transduction processes. Despite the conservation between the rho and ras families, preliminarily experiments have shown that rho and ras function in distinct biochemical pathways. Therefore, rho and ras proteins appear to utilize common functions to achieve different physiological effects. The overall goal of this project is to determine the function of rho proteins and to evaluate the functional similarities and differences between the rho and ras gene families. The yeast gene RHO1 will be the initial subject of the studies, due to preliminary results indicating its involvement in cellular response to the environment. Two complementary approaches will be used simultaneously to investigate RHO1 function. First, the protein will be purified and biochemically characterized in vitro. The eventual aim of these studies is the reconstruction in vitro of a functional RHO1 signal transduction system. The second approach will use the techniques available for isolating specific regions of the yeast genome, engineering this material to desired specifications, and reinserting the altered DNA into the chromosome. This approach will seek to construct an in vivo system for assay of RHO1 function, through the use of conditional-lethal alleles. The genetic approach will also investigate biochemical pathways with which RHO1 interacts, through the identification and isolation of second-site suppressor mutations that modulate RHO1 function. These genetic experiments will complement the biochemical studies by indicating specific RHO1 activities to be tested in vitro. rho genes will be examined for function in heterologous systems, thus revealing whether the family of proteins has been functionally conserved in evolution. In addition to this general investigation of rho activity, the functional relationship of specific regions of rho and ras proteins will be examined by construction of chimeric genes containing domains from both divisions of the ras-rho superfamily.

This award provides funds to aid in the purchase of a variety of equipment items to be used for biotechnology research by members of the zoology and biochemistry departments at Iowa State University. Equipment includes a state of the art imaging device for detection and analysis of radioactively-labelled nucleic acids and proteins, a device for densitometry of stained-gels and autoradiograms, equipment for synthesis and separation of DNA oligomers, a computer workstation for data analysis and several other items. The equipment will be housed in the nucleic acid facility of the new Biotechnology Center. Advances in basic research in Biotechnology, including studies in molecular biology and cell biology, have depended heavily on the development of instrumentation such as DNA synthesizers and sequencers that permit what had been time-consuming and difficult experimentation to be accomplished rapidly and with little effort. New devices have now increased the speed and sensitivity of detection and analysis of proteins and nucleic acids by orders of magnitude. Typically such equipment is placed in a central location where it is available to a large number of investigators who can benefit from its use. Precisely such an arrangement is proposed for the equipment to be purchased through this award.

Adsorption is under development worldwide as a chemical- engineering unit operation for the separation and purification of gaseous and liquid mixtures. In addition to separation of air into its components and removal of pollutants from air and water streams, adsorption is being considered for a host of new applications, including separation of proteins from bioreactor product streams, recovery of carbon dioxide from combustion of fossil fuels, recovery of uranium from sea water, methane storage, and ultrapurification of water and raw materials for the electronics industry. Current research using molecular simulation by grand canonical Monte Carlo and molecular dynamics is providing new insights into the physics of adsorption. Results from these fundamental investigations provide a solid foundation for the development of new theories to replace the classical thermodynamic methods. The objective of this research is to bridge the gap between adsorption theory and practice by developing a molecular theory capable of predicting preferential adsorption from fluid mixtures. Adsorption isotherms of single gases and their binary mixtures, and isosteric heats of adsorption of single gases, will be measured. Experimental data collected for four binary systems will serve to test theories for predicting mixed-gas adsorption equilibria from single component isotherms and heats of adsorption.

Myers 9319028 This project is investigating the molecular mechanisms controlling morphologic development. Multicellular eucaryotes contain many types of genetically identical yet morphologically distinct cells, indicating morphologic differentiation is an integral aspect of development. Little information is available regarding the molecular mechanisms by which morphologic differentiation is regulated. This question will be addressed through the identification of gene loci that influence a defined morphologic differentiation pathway in the ascomycete Saccharomyces cerevisiae. A screen is being used to recognize genes within this group likely to code for regulatory components of the differentiation response. The genes will be characterize by molecular cloning methods, and their sites of action will be ordered in a presumed hierarchy of events that determine the differentiation state. The first specific objective of this project is to identify additional loci in the S. cerevisiae genome required for normal control of cellular dimorphism. Genetic analysis using visual characterization of cell and colony morphology as a scorable phenotype will accomplish this goal. The second objective is to isolate selected genes by molecular cloning, and to determine the primary structure of the corresponding protein products by nucleotide sequence analysis. This goal will be met by selecting plasmids from a genomic library based on their ability to restore yeast-like growth to a specific pseudohyphal mutant. The role of each cloned gene in determination of morphologic differentiation will be confirmed by site-specific mutagenesis and gene replacement methods. The third objective is to determine whether the genes act in a linear pathway controlling the differentiation response and, if so, to determine their relative sites of action. This goal will be accomplished by determining epistasis relationships between genetic elements that either stimulate or repress pseudohyphal growth. %%% Accomplishing these objectives will provide significant new insights into the molecular mechanisms that control development of cell shape. Specific proteins will be identified that control morphologic differentiation, and their primary structures most likely will suggest biochemical activities by which this regulation is accomplished. Information regarding the relative sites of action of these molecules in a differentiation pathway will suggest the function of specific factors. Future investigations could then utilize this information to characterize control of morphologic development at the biochemical level, and to examine whether similar mechanisms are broadly conserved in evolution. ***

The molecular and physiological functions of the Arabidopsis starch metabolism gene network will be determined. The glucose polymers that make up starch, despite their simple chemical structures, display a complex molecular architecture that is essential for starch function. The Arabidopsis genome sequence allows identification of all the genes involved in starch biosynthesis or mobilization, and determination of the function of each gene product individually and within the metabolic network. This aspect of metabolism is a distinguishing feature of all plant life, by which the metabolic gains of photosynthesis are stored and used later when light is not available as the energy source. Understanding starch assembly and disassembly as a comprehensive chemical system, therefore, is required for a complete functional description of how the sequence information in the Arabidopsis genome is translated into the plant life form.
In this project, "determination of function" implies that the role of each protein in the assembly or disassembly of starch polymers will be understood at the level of specific molecular interactions. The project focuses on 28 genes that are likely to be involved in starch metabolism after the production of the glucosyl unit donor. The gene set includes starch synthases, branching enzymes, debranching enzymes, a-amylases, b-amylases, disproportionating enzymes, and starch phosphorylases. In each instance the genome sequence predicts multiple isoforms. The two organizing hypotheses of the project are that most isoforms have specific, non-overlapping roles in creating or dismantling the molecular architecture of starch, and that many components of the network act via direct functional interactions rather than in a series of independent enzymatic steps.
Results will be shared through scientific publication, regular web postings (www.starchmetnet.com), and via scientific conferences. Resources that will be developed and made available to the scientific community include mutant lines, isoform-specific antibodies, and purified recombinant enzymes.
The project will have broader impacts in the training of scientists at all levels in areas such as mRNA profiling, biochemistry, and bioinformatics. The project will strive to involve members of under-represented groups in this activity at the undergraduate level, through established partnerships with universities that traditionally serve such students. Starch provides the majority of calories in the human diet, and is also a renewable resource for energy production and industrial raw materials. Comprehensive understanding of this energy storage system will provide a greater ability to exploit renewable plant resources to meet the continually increasing demands of our changing society and environment.

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Abstract for Award no. 1517256 (MCB-SSB)

This project will develop and test predictive mathematical models of the chemical reactions (metabolism) by which maize (corn) transforms the sugars produced during photosynthesis into compounds such as starch that are stored in the seeds. The goal is to understand starch metabolism better and provide the means for enhancing food production. This study will reveal fundamental principles of metabolic function and dynamics, and provide interdisciplinary training (in mathematical modeling, genetic engineering and metabolic biochemistry) to students engaged in this project.

Starchy endosperm (SE) is the major site of carbohydrate and protein storage in cereals, but atypical constraints complicate detailed resolution of the system. These include a steady state oxygen level of essentially zero, coincident with energy-expensive breakdown and re-synthesis of building blocks prior to storage polymer formation. A major question in SE metabolism is how ATP is generated to support starch and protein synthesis, and how the photo-assimilate is divided between catabolic and anabolic pathways. This project will expand upon existing flux models to account for bio-energetic parameters, and probe the system by directed genetic modifications that impair specific metabolic nodes. Metabolite levels and flux maps will be compared between SE from unaltered and mutant lines. These data will be applied to refine the mathematical models and test hypotheses of how the system diverts chemical resources for storage compound accumulation.