Thomas Peterson - US grants

Previous studies in the dog have demonstrated that intravascular mechanoreceptors have effects on renal excretory function via reflex modulation of arginine vasopressin, renin and catecholamine levels and efferent renal nerve activity. These afferent neural pathways are thus believed to provide the link between changes in blood volume and appropriate changes in salt and water excretion. It has been further hypothesized that a depressed sensitivity or resetting of these receptors may contribute to the fluid retention seen in heart failure and that the efficacy of cardiac glycoside treatment in this condition may be partially due to this treatment increasing the sensitivity of these receptor mechanisms. However, the role of these neural mechanisms in maintaining circulatory and body fluid homeostasis in the primate is still unclear. Studies using the primate model have demonstrated that afferent neural control of renal function in this species is less sensitive than in the dog and may be linked more to control of blood pressure than control of blood volume. Furthermore, it has been hypothesized that, unlike the dog, the renal nerves may not play a major role in directly affecting renal sodium reabsorption in the primate which suggests that efferent neural control of renal excretion may be less important in this species also. The studies in this proposal will extend these observations and more completely explore the possible importance of these neural mechanisms in the nonhuman primate. The specific aims are: 1) to determine if acute and chronic renal denervation affect the antinatriuretic response to orthostasis and if chronic renal denervation causes the kidney to become hypersensitive to circulating norepinephrine, 2) to determine if there is interaction between atrial receptor and carotid sinus baroreceptor control of renal function and if cardiac glycoside treatment sensitizes these effects, (3) to determine if cardiac glycosides affect vagal cardiopulmonary baroreceptor and sinoaortic baroreceptor control of renal nerve activity during changes in blood volume. (4) To determine if vagal afferent pathways have tonic effects on arginine vasopressin, renin and catecholamine levels and baroreceptor interaction with these effects, (5) to directly determine the relative importance of cardiac receptors and baroreceptors in modulating the secretion of vasopressin, renin and catecholamines during changes in central blood volume and (6) to determine if cardiac receptors and/or baroreceptors are responsible for the interaction between volumetric and osmotic control of vasopressin.

Previous studies in the dog have demonstrated that intravascular mechanoreceptors have effects on renal excretory function via reflex modulation of arginine vasopressin, renin and catecholamine levels and efferent renal nerve activity. These afferent neural pathways are thus believed to provide the link between changes in blood volume and appropriate changes in salt and water excretion. It has been further hypothesized that a depressed sensitivity or resetting of these receptors may contribute to the fluid retention seen in heart failure and that the efficacy of cardiac glycoside treatment in this condition may be partially due to this treatment increasing the sensitivity of these receptor mechanisms. However, the role of these neural mechanisms in maintaining circulatory and body fluid homeostasis in the primate is still unclear. Studies using the primate model have demonstrated that afferent neural control of renal function in this species is less sensitive than in the dog and may be linked more to control of blood pressure than control of blood volume. Furthermore, it has been hypothesized that, unlike the dog, the renal nerves may not play a major role in directly affecting renal sodium reabsorption in the primate which suggests that efferent neural control of renal excretion may be less important in this species also. The studies in this proposal will extend these observations and more completely explore the possible importance of these neural mechanisms in the nonhuman primate. The specific aims are: 1) to determine if acute and chronic renal denervation affect the antinatriuretic response to orthostasis and if chronic renal denervation causes the kidney to become hypersensitive to circulating norepinephrine, (2) to determine if there is interaction between atrial receptor and carotid sinus baroreceptor control of renal function and if cardiac glycoside treatment sensitizes these effects, (3) to determine if cardiac glycosides affect vagal cardiopulmonary baroreceptor and sinoaortic baroreceptor control of renal nerve activity during changes in blood volume. (4) to determine if vagal afferent pathways have tonic effects on arginine vasopressin, renin and catecholamine levels and baroreceptor interaction with these effects, (5) to directly determine the relative importance of cardiac receptors and baroreceptors in modulating the secretion of vasopressin, renin and catecholamines during changes in central blood volume and (6) to determine if cardiac receptors and/or baroreceptors are responsible for the interaction beteen volumetric and osmotic control of vasopressin.

Changes in renal salt and water excretion secondary to alterations in blood volume represent appropriate responses for maintaining body fluid homeostasis. In spite of the simplicity of the concept, the mechanisms linking blood volume and renal excretion are not clearly understood. This is particularly true for the nonhuman primate since little investigative work has been done using this species, especially experiments on conscious animals. The primary objective of the research proposed in this application is to clarify and better define the functional impartance of some of these blood volume control mechanisms in the conscious monkey. The factors to be investigated include renal nerves, cardiac nerves and atrial natriuretic factor (ANF). Thus the objective encompasses both neural and humoral mechanisms. The specific aims are: (1) to determine if the renal nerves exert tonic or basal effects on renal excretion, including assessments of the effect of unilateral renal denervation on contralateral innervated kidney function and potential importance of denervation hypersensitivity to norepinephrine as a factor which may complicate the interpretation of chronic renal denervation protocols, (2) to determine the necessity of the renal nerves and/or increases in ANF for eliciting postprandial increases in urinary excretion, (3) to determine the necessity of the renal nerves and/or increases in ANF for eliciting the renal excretory responses to chronic changes in dietary sodium intake, (4) to determine if the level of sodium intake affects the magnitude of and renal nerve and ANF contribution to the renal responses to volume expansion, (5) to determine the sensitivity of the renal and humoral responses to increases and decreases in blood volume and necessity of the renal nerves for eliciting the excretory effects and (6) to determine the role of cardiac receptors in eliciting changes in plasma vasopressin, ANF and norepinephrine levels and plasma renin activity during graded changes in central blood volume (lower body positive and negative pressure). The results from these experiments may suggest possible mechanisms which may be involved in the pathogenesis of congestive heart failure and hypertension, conditions often characterized by body fluid imbalance.

ABSTRACT CTS-9512541 A three-component fiber optic Phase Doppler Particle Analyzer/Laser Doppler Velocimeter (PDPA/LDV) which will be acquired for non-intrusive measurements of two-phase flow. The PDPA/LDV system will enhance current research projects at the university, and will provide opportunties for a multitude of new collaborative and interdisciplinary efforts. Research programs include gas-solid flows and the modulation of gas-phase turbulence in the presence of particles, turbulence studies using Planar Laser Induced Fluorescnence, combustion research and turbulent mixing phenomena, chemigation research, and flow characterization in plasma etch systems. ***

Plant genomes are laden with local sequence duplications and clusters of homologous genes. To simplify the analysis of these duplications and gene clusters, this project will develop a new genetic technology to generate chromosomal deletions quickly and efficiently. This approach is based on the finding that transposable elements can participate in alternative transposition pathways that generate novel recombination products, including large deletions and duplications. The system described here utilizes a transgene construct containing maize Ac/Ds transposon ends in tandem orientation within a I/dSpm transposon (Ned1; Nested deletions 1). The Ned1 construct will be transformed into maize; subsequent crosses will introduce the En/Spm transposase to mobilize Ned1 to various genomic locations, and the Ac transposase to activate the deletion process. The action of Ac transposase on the Ac termini within Ned1 generates an unlimited set of nested deletions with one end anchored at the transgene locus. The Ned1 construct contains marker genes for detection of both Ned1 transpositions and Ac--induced deletions, as well as sequences for easy cloning of deletion endpoints via plasmid rescue. The expected outcome of this project is the production of maize plants containing the Ned1 transgene construct. These plants and their progeny will be tested for 1) transposition of Ned1 by En/Spm transposase; 2) induction of deletions by Ac transposase; 3) transmission of deletions to progeny plants. Additionally, the Ned1 transgenic lines, En/Spm- and Ac-containing lines will be backcrossed to a commonly-used maize inbred line [B73] in order to make these materials most useful for the research community. This approach could be extended to the production of a set of maize lines containing Ned1 elements at dispersed sites throughout the genome that could be used to isolate deletions and other rearrangements in specific regions of the genome.
Deliverables:
1. This project will generate maize plants containing the Ned1 transgene construct.
2. Ned1 transgenic plants and their progeny will be tested for a) transposition of Ned1 induced by En/Spm transposase; b) formation of deletions induced by Ac transposase; c) transmission of deletions to progeny plants.
3. Ned1 transgenic lines, En/Spm- and Ac-containing lines will be backcrossed to a commonly-used maize inbred line [B73] in order to make these materials most useful to the research community.

Contact Information for Deliverables:
Thomas Peterson, 2206 Molecular Biology, Iowa State University, Ames, IA 50011
thomasp@iastate.edu

The goal of this project is to develop a method for efficient homology-dependent site-specific recombination (SSR) in Arabidopsis. The approach used is based on the recombination-inducing properties of transposable elements, coupled with alternative methods of delivering recombination substrates to plant cells. In previous research, the PI and co-PI have shown that DNA and RNA transposable elements can stimulate recombination. First, excision of maize Ac/Ds DNA transposons greatly induces homologous recombination in plants, including Arabidopsis. Second, retrotransposons can generate high levels of cDNA that recombine readily with genomic sequences. This project will develop two-component (recipient and donor) transgene constructs that contain partially-overlapping visible and selectable marker gene fragments. The recipient component contains, in addition, a maize Ds element inserted between the marker genes. The recipient construct is integrated into the Arabidopsis genome; upon expression of Ac/Ds transposase, Ds excision generates a recombination hotspot in the recipient construct. The donor construct sequences are delivered into the plant cells via several alternative methods, including Agrobacterium T-DNA transformation, particle bombardment, ectopic chromosomal position, and retrotransposon-generated cDNA. Recombination of recipient and donor sequences can be detected in somatic cells by the visible marker, and events transmitted to progeny can be genetically selected. Heritable recombination events will be characterized by molecular and genetic analysis to gain a greater understanding of SSR mechanisms in plant cells. Project results will be available at the following web site: . All reagents (seeds of transformed lines, plasmid vectors) will be made available upon request.

Homology-dependent recombination is a powerful tool for genetic modification in many organisms; however, methods for routine SSR are not yet available for higher plants. An explicit goal of the Arabidopsis 2010 project is the development of methods for directed mutations and SSR. Successful completion of this project will provide an important new means for making precise changes in plant genomes for both fundamental research and practical applications.

Partial support is requested for a conference on inTransposition, Recombination and
Applications to Plant Genomicslo, to be held from June 5 to 8, 2003 at Iowa State University, Ames,
Iowa. The meeting will include presentations by 18 speakers who are leaders in different areas of
transposable element and recombination research. Up to nine additional speakers will be selected from submitted abstracts for short oral presentations. The meeting also includes poster sessions and time for informal discussions. The conference incorporates programmatic and educational features to encourage meaningful participation by scientists and students representing diversity in experimental system, career level, ethnicity and gender.

Collaborative Research: Comprehensive Molecular, Genetic and Cytogenetic Analysis of Transposon-Induced Chromosomal Rearrangements in Maize

Transposons are segments of DNA that can move (transpose) from one site to another in the genome. Sometimes the ends of separate transposons will attempt to transpose; this alternative transposition reaction can cause large rearrangements of chromosomes. The aims of this project are to determine which arrangements of transposons can give rise to alternative transposition reactions, and what kinds of chromosomal aberrations can be produced. The research will be conducted using maize (corn), an ideal model system in which transposition events can be detected by changes in kernel color. The results should be relevant to other organisms, including humans. The results of this project will help to elucidate basic principles of the biology of transposable elements and their impact on genome structure and expression: First, this research will lead to a greater understanding of the potential role of transposable elements in genome diversification and speciation. Second, the results could help to explain the origin of spontaneous chromosomal aberrations that are often associated with certain human genetic diseases including cancer. Third, the results may provide the basis for development of new methods for studying eukaryotic chromosomes. An important aspect of this project is the use of complementary approaches to bridge the molecular and chromosomal levels of structure. In addition, students will be trained in methods of chromosome analysis through the research project and workshops offered by the investigators.

The next challenge in plant biology is to discern the function of the many genes revealed through the various genome sequencing projects. One important approach for understanding gene function is to study the consequence of removing or knocking out the function of a specific gene. In the model plant Arabidopsis, efficient methods are available to knock out many genes of interest. Functional analysis, however, is often frustrated by genetic redundancy or the presence of multiple copies of the same or closely related genes. Particularly prevalent in plants are tandem gene duplications, and as many as 15% of Arabidopsis genes are organized in tandem arrays. A targeted mutagenesis protocol will be developed to augment existing approaches for understanding Arabidopsis gene function, particularly for genes for which functional analysis has been confounded by genetic redundancy. The approach uses zinc finger nucleases (ZFNs) - chimeric proteins made up of a zinc finger array fused to the DNA cleavage domain of Fokl endonuclease. Zinc finger arrays of high affinity and specificity can be engineered to recognize virtually any target gene. Upon cleavage of the target DNA by the ZFN, the broken ends are repaired inefficiently, resulting in locus-specific mutations. In initial experiments, a model zinc finger nuclease and a reporter gene containing the cognate target site will be used to optimize methods for efficient recovery of heritable ZFN-induced mutations. Next, ZFNs will be engineered to recognized native Arabidopsis loci, including those that give observable phenotypes when mutated. ZFNs will also be engineered for a locus where mutatations do not result in an observable phenotype. To recover mutations at such a locus, robust DNA amplification and sequence-based detection methods will be employed. Lastly, a tandem array of duplicate genes will be targeted. Deletion of the array will be accomplished by engineering ZFNs to cleave within the outermost genes. The frequency of recovering deletions and individual mutations will be determined to assess the relative efficiency of this approach. In addition to Arabidopsis, this mutagenesis approach will also be valuable for plant species such as rice and maize, where genome sequencing projects are ongoing and tandem genes are abundant. Moreover, the ability to generate and regulate specific chromosomal double-strand breaks will facilitate studies of chromosome structure and DNA repair mechanisms in plants. The URL for the web site where protocols as well as results of gene knock-out studies can be accessed is http://www.beckmancenter.umn.edu/html/2010.html.

Understanding the function of plant genes is critical if plants are to be fully harnessed to meet the world's burgeoning need for fuel, feed and industrial raw materials. Although significant progress has been made in discerning plant gene function, existing approaches have inherent limitations, particularly with respect to ascribing function to redundant or duplicated genes. The goal of this project is to implement an efficient targeted mutagenesis approach to overcome these limitations. This project will train undergraduate interns and graduate students for work in plant molecular biology. Students from underrepresented groups will be recruited to the project through programs designed to enhance minority participation in science.

Research Objectives: Partial chromosome duplications (segmental duplications) are important contributors to the structure, function and diversity of plant and animal genomes. However, very little is known concerning how duplications are generated, and their immediate effects on gene expression and genetic recombination. This project examines the potential role of transposable elements, or jumping genes, in generating segmental duplications. The aim of this project is to isolate and characterize a series of partial chromosome duplications in corn generated by Ac/Ds transposable elements. The structures of duplications will be examined by microscopic visualization of chromosomes. The duplication endpoints will be isolated and studied to determine precisely how DNA replication and genetic recombination are involved in forming duplications. Finally, the effects of partial chromosome duplications on genetic recombination and gene expression will be analyzed in detail, because these two processes are critical for effective plant breeding. This project utilizes complementary molecular, genetic and microscopic approaches to study this important class of genetic variants.

Broader Impacts: This project will provide significant new insight into the role of transposable elements in generating duplications, and the impact these duplications have on genetic recombination and gene expression. This information may lead to significant advances in breeding of crop plants, thereby improving agricultural efficiency and environmental sustainability. In addition, this project will result in significant training opportunities for current and future scientists in a variety of approaches and methods in molecular genetics, classical genetics, cytogenetics and transposon biology. Three graduate students are integral to the research project and will be trained in a variety of classical, cytological, and molecular genetic approaches. The project will also provide laboratory work experience and on the job training for approximately six undergraduate students per year. In addition, an annual cytogenetics short course will train 12-18 high school students and teachers yearly. Together these training activities will significantly enhance the science infrastructure in the USA.