Phillip B. Danielson, Jr. - US grants

Fogleman 9806888 Cytochrome P450 genes produce enzymes that allow insects to tolerate poisonous chemicals such as insecticides and certain plant compounds. Some plants protect themselves from insects (and other herbivores) by making chemical compounds that are toxic to the animals which try to feed on them. Cytochrome P450 enzymes detoxify chemical compounds, usually by changing their chemical structure. The study of P450 genes, therefore, will provide information as to how insects become resistant to insecticides and/or plant defensive chemicals. This proposal involves the use of an insect-plant model system. The insects are fruit flies of the genus Drosophila, and the plants are columnar cacti which grow in the Sonoran Desert of southwestern United States and Mexico. A wealth of background information on this model system has already been obtained including data on the ecology and genetics of the fruit flies and identification and characterization of the toxic chemicals that the cacti produce. Employing modern techniques of molecular genetics, previous experiments have isolated over 100 P450 genes from desertadapted drosophilids. Since there are multiple P450 genes in each insect species, one of the most important and currently unanswered questions is what is the relative contribution of individual P450 genes to resistance? The experiments included in this proposal will investigate this question and will significantly contribute to our understanding of the mechanisms by which insects evolve P450-mediated resistance. This objective represents a major advancement in the areas of insect ecological genetics and evolution.

PROJECT SUMMARY Robert Dores Gene duplication is a recurring theme in the evolution of vertebrate polypeptide hormones and neuropeptides. These duplication events lead to the formation of gene families in which divergence of function is the usual outcome. In the case of the opioid-coding genes, duplication events have proceeded along two paths: a) an apparent duplication of function (both Proenkephalin and Prodynorphin circuits function as inhibitory networks in the central nervous system); or b) divergence of function as seen in analgesic activity of Proenkephalin and Prodynorphin products, as compared to the heightened pain responsiveness (nociceptic) activity of Pronociceptin products, or the melanocortin (color change and chronic stress regulation) activity of Proopiomelanocortin products. Are these duplication events entirely random, or do they correspond to discrete points of radiation of the vertebrates? This proposal develops the hypothesis that the duplication-driven expansion of the opioid-coding gene family (Proenkephalin, Prodynorphin, Pronociceptin, and Proopiomelanocortin) corresponds to periods when polyploidization (replication of the entire genome) altered the course of vertebrate evolution. To identify these "burst" periods, Dr Dores and colleagues have combined a comparative approach with a molecular approach. The species selected for this analysis (lungfish, urodele amphibians, and a ancient lineage of anuran amphibians) represent lineages that bracketed one of the predicted genome duplication events (the rise of the lobed finned fish and tetrapods in the Devonian). Thus, by taking a comparative approach it is possible to select taxa that fit into a logical phylogenetic hypothesis based on the fossil record. By employing the molecular approach, it is possible to test that hypothesis. In this study, the opioid-coding gene family will be used as a model to analyze how natural selection acts on duplicated genes to alter sequence, and potenti ally alter function. The key to the molecular approach is to focus on an ancestral character common to all members of this gene family - the opioid core sequence YGGF(M/L). By taking this approach, we have already detected genes in ray-finned fish and lobe finned fish (previous period of support) which were previously unsuspected. In addition, novel opioid peptides have been revealed whose opiate agonist potential has not been evaluated. While it is appreciated that none of the taxa used in this study are a "living fossil," by taking a comparative/molecular approach it is possible to reconstruct, through cladistic paradigms (maximum parsimony), the most likely pathways which have lead to the extant opioid-coding genes, and the potential ramifications of these genes with respect to evolution of neuronal circuits in the vertebrate central nervous system.

AWARD ABSTRACT - 9977691

This award will fund the purchase of a Beckman Coulter CEQ 2000
Automated DNA Sequencing and Sample Prep Core Unit. Based on
well-established DNA sequencing technology, this system will provide for a
significant increase in the productivity and cost effectiveness of
undergraduate/graduate research and education in molecular biology at the
University of Denver. Specific research programs that will benefit
immediately include National Science Foundation-funded studies on: the
molecular evolution of endocrine neuropeptide hormones, avian sex
chromosomes, and insect steroidogenesis; the population genetics of
endangered species; the use of site-directed mutagenesis to delineate the
structural properties of proteins; and the regulation and ecological
significance of the cytochrome P450 superfamily of toxin-metabolizing
enzymes. Additionally, use of the automated sequencer in collaborative
work in conservation genetics with researchers from the Denver zoological
gardens, will further studies of the genetic diversity of endangered and
threatened species.
Beyond the in-depth training of undergraduate and graduate
students in the research laboratory setting, a broad range of
classroom-oriented educational goals will be advanced. Benefits will be
particularly evident in the molecular-oriented laboratory courses that are
at the heart of the new Bachelor of Science and Bachelor of Arts degrees
in Molecular Biology offered by the Department of Biological Sciences.
Department-sponsored biotechnology classes offered to high school students
and teacher-training workshops that promote hands-on science education at
the secondary school level will also be greatly enhanced. The benefit to
high school outreach efforts is immeasurable given that these programs
target students in urban and low-income school districts who have
traditionally been underrepresented in the natural sciences.
In short, acquisition of the CEQ 2000 DNA Sequencing and Sample
Prep Core Unit will provide significant and immediate benefits to
education at the secondary, undergraduate and graduate levels, while
providing a cost-efficient means of satisfying the growing DNA sequencing
needs of researchers funded by the National Science Foundation in both the
Department of Biological Sciences and the Department of Chemistry and
Biochemistry.

Award Abstract
A grant has been awarded to Dr. Phillip Danielson at the University of
Denver to fund the purchase of a real-time quantitative PCR System. Based on well-established laser fluorescence and DNA amplification technology, this system will significantly increase the productivity and cost effectiveness of undergraduate/graduate research and education in molecular biology. Specific research programs that will benefit immediately include National Science Foundation-funded studies on: (1) endocrine hormones in the brain that are responsible for pain and stress responses in living organisms; (2) cytochrome P450 toxin-metabolizing enzymes - an understanding of which is critical to the control of agricultural pests and many disease-carrying organisms and; (3) the genetic diversity of endangered and threatened species which the University of Denver conducts in collaboration with the Denver Zoological Gardens and the Denver Museum of Science and Nature.
Until recently, the measurement of gene expression using traditional assays has meant numerous rounds of laborious optimization, test template dilutions and post assay manipulations. Even then, the estimated concentration of a genetic message was often inaccurate owing to the unpredictable variability of traditional endpoint-based measurements. This is because small biases in amplification efficiency over the course of an assay would produce large differences in the amount of final product being measured. A solution to these problems was found in the new generation of real-time quantitative PCR Systems. The system monitors reaction kinetics in real-time making it possible to quantitate DNA and RNA concentrations in the smallest of tissue samples with unparalleled accuracy, precision and speed. An added benefit is that the PCR instrument can also be used for high-speed genotyping. The automated liquid handling capabilities already in place at the University of Denver will handle sample preparation to ensure run-to-run consistency by minimizing pipetting errors and crossover contamination.
Beyond the benefit to the research activities of faculty at the University of Denver, a broad range of laboratory and classroom-oriented educational goals will be advanced at both the undergraduate and graduate levels. Benefits will be particularly evident in the molecular-oriented laboratory courses that are at the heart of the new Bachelor of Science and Bachelor of Arts degrees in Molecular Biology offered by the Department of Biological Sciences. On a broader level, department-sponsored biotechnology classes offered to high school students and teacher-training workshops that promote hands-on science education at the secondary school level will also be greatly enhanced. The benefit to high school outreach efforts is immeasurable given that these programs target students in urban and low-income school districts who have traditionally been underrepresented in the natural sciences. In short, acquisition of the PCR System will provide significant and immediate benefits to education at the secondary, undergraduate and graduate levels, while providing a cost-efficient means of satisfying the growing DNA analysis needs of life-science researchers funded by the National Science Foundation.

A grant has been awarded to Dr. Phillip Danielson at the University of Denver to fund the purchase of a Transgenomic Nucleic Acid Fragment Analysis System. Based on established DNA size separation and mutation detection technology, this system will increase the quality and cost effectiveness of undergraduate/graduate research and
education in molecular biology. Specific research programs that will benefit immediately include National Science Foundation-funded studies to identify novel genes that encode: (1) endocrine hormones in the brain; (2) cytochrome P450 toxin-metabolizing enzymes - which are critical to the control of crop pests and disease-carrying organisms; 3) microbial proteins that can be used to clean up of toxic waste sites contaminated with heavy metals and 4) molecular markers that can be used to identify and track genetic diversity in endangered species - work is conducted in collaboration with the Denver Zoological Gardens and Denver Museum of Science and Nature.

Until recently, the identification of mutations required the laborious screening of hundreds to thousands of genes for subtle variations in DNA sequence. Analysis of a single novel gene by the direct sequence approach currently used, can require a day or more to complete. The Transgenomic WAVE System funded by this grant will reduce the analysis time to 2-4 minutes/sample. The discovery and analysis of genes that encode proteins involved in toxin breakdown, as well as neuropeptides linked to stress is the focus of several research programs. Since these genes often exist as duplicates with subtle but critical differences, it is essential that both copies be isolated. The WAVE system will be used to reduce the potential number of competing non-target gene fragments by precise size fractionation of the initial pool of DNA used for gene amplification reactions. The instrument's mutation detection and fragment capture functions will be used to increase the efficiency with which these related genes are identified and recovered - even where two genes sequences differ by less than 0.5%. In research focused on conservation biology and microbial ecology, the WAVE system will provide an extremely sensitive approach to the analysis of DNA sequence differences among and within species. Finally, the WAVE's high-speed genotyping capabilities will be used to gather gene frequency data from hundreds of samples for large-scale, multi-state conservation genetic projects.

Beyond the benefit to the research activities at the University of Denver, a broad range of
laboratory and classroom-oriented educational goals will be advanced at both the undergraduate and graduate levels. Benefits will be particularly evident in the molecular-oriented laboratory courses that are at the heart of the Bachelor of Science and Bachelor of Arts degrees in Molecular Biology. On a broader level, department-sponsored biotechnology classes offered to high school students and teacher-training workshops that promote hands-on science education at the secondary school level will also be greatly enhanced by providing first-hand experience in one of the most modern methods of genetic analysis. The benefit to high school outreach efforts will immeasurable given that these programs target students in urban and low-income school districts who have traditionally been underrepresented in the natural sciences. In short, acquisition of the
WAVE Nucleic Acid Fragment Analysis System will provide significant and immediate
benefits to education at the high school, undergraduate and graduate levels.

A striking feature of chemical communication systems in the brains of jawed vertebrates (gnathostomes) is the apparent duplication of genes that produce useful neuropeptide compounds. For example, in mammals there are four neuropeptide precursors (proenkephalin, prodynorphin, proopiomelanocortin, and proorphanin) that produce opiate-like peptides (opioids) or nociceptic-like peptides (orphanins). These four precursors are members of a gene family, the opioid/orphanin family, and the apparent redundancy of genes in this family in fact reflects the evolutionary history of the vertebrates. Gene duplication events at discrete points in vertebrate evolution have allowed for the layering of neuropeptide networks over time, and have led to a diversification in function of these peptide products, including roles in analgesia, nociception, motor control, and feeding. This project combines molecular biology procedures with a comparative approach to define trends in the evolutionary radiation of the orphanin/opioid gene family in gnathostomes. The cloning and sequencing of cDNAs, coupled with the measurement of gene expression by real-time PCR, will generate a database of sequences for each gene in the family that will be used to perform cladistic analyses and to identify novel opioid sequences. By examining selected vertebrate lineages where the evolution of this gene family has been slower than in mammals or teleost fish, it is possible to make inferences about the origin and transitions in the sequential evolution of this gene family. These data will test the 'proenkephalin hypothesis' that the ancestral gene in the opioid/orphanin gene family was a gene that coded for the enkephalin-like product. Results will provide a new level of understanding of the evolutionary mechanisms underlying the functional diversity in this important family of neuropeptides.
The impact of this project will extend beyond neuroendocrinology to molecular neurobiology in general, and to evolutionary biology with respect to the trends that promote species diversity. Multi-disciplinary undergraduate and graduate training also is an important component of this project.

DESCRIPTION: (Provided By Applicant) The hypothalamus-pituitary-adrenal (interrenal) axis HPA(l)J is a neuroendocrine network responsible for modulating a broad range of physiological functions from reproductive activity to chronic stress response. Within this network, corticotropic cells in the anterior lobe of the pituitary express proopiomelanocortin (POMC), a precursor protein from which the polypeptide hormone, adrenocorticotropin (ACTH), is post-translationally released. ACTH is well established as a critical link in this network. Neurons in the hypothalamus secrete corticotropin releasing hormone (CRH), which induces the secretion of ACTH from the anterior pituitary. ACTH, in turn, stimulates the adrenal gland to synthesize and release cortisol, which is the final hormone in the chronic stress response cascade. Fluctuations in the production and/or regulation of CRF, ACTH or cortisol can have serious consequences with respect to the survival of an organism. Hyposecretion of cortisol results in Addison's Disease. Conversely, hypersecretion of cortisol is associated with Cushing's Syndrome, a multi-symptom metabolic disorder characterized by muscle atrophy, immune deficiency, adrenal hyperplasia, kidney dysfunction and general tissue degeneration. In contrast to humans, where the U.S. frequency of Cushing's Syndrome is on the order of 3700 cases annually, 100 percent of Pacific salmon display Cushing's Syndrome-like tissue and organ degeneration coincident with spawning. While it had been thought that the stress of marine to freshwater migration was responsible for the post-spawning demise of these fish, studies have now implicated overproduction of cortisol during sexual maturation as the factor which ultimately leads to the demise of spawning salmon. The current proposal will use a combination of cell and molecular strategies to investigate the role of P0MG and hypothalamic neuropeptides (CRH, AVT and Uro I) in the regulation of the HPA axis. This wilt help to identify specific components of HPA(I) axis which are altered during the sexual maturation of Pacific salmon, and which lead to their inevitable post-spawning demise. An understanding of the molecular mechanisms underlying the development of Cushing's Syndrome-like pathology in Pacific Salmonids has the potential not only to advance our knowledge of the role of the HPA(I) in reproductive stress, but to contribute to a broader understanding of the etiology and pathogenesis of hypercorticism in humans.

With support from the Chemistry Research Instrumentation and Facilities (CRIF) Program, the Department of Chemistry at the University of Denver will acquire a Matrix-assisted Laser Desorption Ionization - Time of Flight (MALDI-TOF) Mass Spectrometer. This instrument will be used in a wide variety of research projects, including a) structural and thermodynamic properties responsible for the proper/improper protein folding; b) the mechanisms by which halogens cause degradation of polyamide membranes used in municipal water filtration systems; c) photocleavage of molecular scaffolds; and d) posttranslational processing and modification of analgesic neuropeptide hormones involved in the management of pain and stress by the brain.

Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry is the technique of choice for obtaining accurate molecular weights on molecules up to and over 300,000 daltons, with extremely high sensitivity. Use of a MALDI-TOF mass spectrometer has therefore become a standard technique in studies involving biomolecules. At the University of Denver, this instrument will be used not only by graduate students in their research but also by undergraduates in laboratory courses where students work with professors in project-oriented teams that conduct experiments involve the purification and characterization of proteins and other macromolecules.

A grant has been awarded to the University of Denver under the direction of Dr. Phillip Danielson for partial support of the purchase of a Protein Fractionation System. The accurate fractionation, quantization, recovery and characterization of individual proteins from complex proteomes are capabilities that are increasingly essential to the growth and success of biological research and education. Until recently, the analysis of whole proteomes has been heavily dependent on 2-Dimensional Gel Electrophoresis (2DGE)-based approaches. This approach required the laborious screening of hundreds to thousands of resolved "spots" on thin gels. The identification of even a small number of proteins of interest, can require weeks to months to complete. These more traditional methods have several critical shortcomings. 2DGE provides poor resolution of the small peptide hormones and larger membrane-associated proteins that are the focus of many of researchers programs. Furthermore, 2DGE yields results that are often difficult to quantify or reproduce. The Protein Fractionation System provides a cost-effective solution to the traditional limitations of 2DGE-based proteomic research. Test data from difficult samples have confirmed the performance and applicability of the system for our research needs.

Specific research programs that will benefit immediately include NSF-funded studies of the posttranslational processing and modification of neuropeptide hormones involved in the management of reproductive stress and proteomic research aimed at elucidating the underlying neuroendocrine mechanisms of mammalian feeding behavior. Other studies are examining the molecular neurobiology of mammalian taste cells, the recycling of neuroendocrine secretory vesicles and the degradation of misfolded proteins. Finally the Protein Fractionation system will advance collaborative research in molecular ecology and conservation biology conducted in collaboration with the Denver Botanical Gardens. Beyond the benefit to basic research at the University of Denver, a broad range of laboratory and classroom-oriented educational goals will be advanced at both the undergraduate and graduate levels. Benefits will be particularly evident in the molecular-oriented laboratory courses that are at the heart of the Bachelor of Science and Bachelor of Arts degrees in Molecular Biology.

On a broader level, department-sponsored biotechnology classes offered to high school students and teacher-training workshops that promote hands-on science education at the secondary school level will also be greatly enhanced by providing first-hand experience in one of the most modern methods of proteomic analysis. The benefit to high school outreach efforts will be immeasurable given that these programs target students in urban and low-income school districts who have traditionally been underrepresented in the natural sciences. Most importantly, exposing pre-college students to modern technologies will have a significant and positive impact on student excitement about science as a career.