Robert M. Dores - US grants

Pro-dynorphin is the common precursor for three opioid peptides: dynorphin A, dynorphin B, and alpha-neo-endorphin. Although there is considerable information on the anatomical distribution of these peptides, the structure of the precursor, and the sequences of the end products derived from this precursor, relatively little is known about the postnatal ontogeny of this system, the steps involved in the post-translational processing of pro-dynorphin, or the mechanisms for regulating region specific proteolytic processing of pro-dynorphin. This proposal will consider two issues: 1) the postnatal ontogeny of pro-dynorphin in the magnocellular/posterior pituitary system and the striato-nigral system; 2) determine whether postnatal neurons obtained from the hypothalamus and striatum are suitable for in vitro monolayer tissue culture studies. The initial studies will monitor the steady state levels of pro-dynorphin-related end products in the posterior pituitary and substantia nigra during postnatal development. These studies will involve radioimmunoassay analyses in conjunction with gel filtration chromatography and high performance liquid chromatography. The in vitro studies will test the viability of different aged neonatal neurons in monolayer culture with a pulse radiolabeling paradigm. Analyses will be done by immunoaffinity column chromatography in conjunction with SDS polyacrylamide gel electrophoresis.

The polyprotein, pro-opiomelanocortin, the common precursor for ACTH- and beta-LPH-related polypeptides, can undergo a variety of post-translational events to yield distinct sets of end products in different regions of the mammalian pituitary. This biosynthetic pathway is also found in representatives of all the classes of non-mammalian vertebrates, and these systems may provide unique models for studying different strategies for regulating the biosynthesis and post-translational processing of pro-opiomelanocortin (POMC). These studies will investigate the mechanisms for proteolytic cleavage and N-acetylation of POMC-related products in the pituitaries of four diverse species of non-mammalian vertebrates: the reptile, Anolis carolinensis; the amphibian, Xenopus laevis; the primitive bony fish, Amia calva; and the cyclostome, Petromyzon marinus. Steady state analysis of extracts of anterior and intermediate pituitary by radioimmunoassay in conjunction with gel filtration, ion exchange and reverse phase HPLC procedures, and in vitro pulse radiolabeling studies of these tissues in culture will provide a broad phylogenetic view of the strategies for the proteolytic processing and N-acetylation of POMC-related end products. The effects of background adaptation and chronic stress on the POMC pituitary systems will be investigated in A. carolinensis and X. laevis. These studies will use steady state analysis and in vitro biosynthetic labeling procedures to study the effects of these paradigms on changes in the rate of synthesis, patterns of post-translational processing, and the rate of secretion of POMC products. These studies will examine different strategies for the regulation of differential processing of a polyprotein, and will serve as a basis for future projects to gain insights into the mechanisms that have influenced the evolution of the pro-opiomelanocortin biosynthetic pathway.

There is evidence that Sudden Infant Death Syndrome occurs as a result of a dysfunction of the respiratory centers in the central nervous system. There is also evidence that exogenous administration of opioid peptides can exert depressor action on the medullary respiratory centers. Since the medulla oblongata is the site of several distinct sets of neurons which synthesize the opioid precursors pro-opio-melanocortin, pro-enkephalin and pro- dynorphin, the possibility exists that these endogenous opioid peptide systems may modulate the medullary respiratory centers. Research in this area would be aided by studies on animal model systems. This proposal focuses on the pro-opiomelanocortin system in the medulla oblongata of the rat during ontogeny. The objectives of these projects are to determine whether there are developmental changes in the degree of proteolytic processing of pro-opio- melanocortin and the degree of N-acetylation of beta-endorphin during late fetal and neonatal development. The project will be approached in two phases. The first series of experiments will involve a steady state analysis of the forms of pro-opiomelanocortin-related end products in the medulla. Tissue collected at fetal day 16, day 18 and neonatal day 1, 7, 14, and 21 will be separately fractionated by gel filtration and analyzed by series of radioimmunoassays specific for beta-endorphin, alpha- MSH and ACTH(1-39). The degree of N-acetylation of beta- endorphin and alpha-MSH will be determined by ion exchange chromatography and reverse phase HPLC, respectively. The second series of experiments will attempt to study the pro- opiomelanocortin biosynthetic pathway in medullary fetal neurons in culture. Dispersed neurons will be pulsed with tritiated tyrosine and the newly synthesized peptides will be isolated by immunoaffinity chromatography and analyzed by SDS PAGE. These projects will provide insights into the developmental appearance of the pro-opiomelanocortin system in the medulla oblongata. This information will be useful for designing experiments to test the potential involvement of endogenous beta-endorphin in the depression of the medullary respiratory complex.

The pituitary hormone, adrenocorticotropin (ACTH), is derived from the precursor protein, pro-opiomelanocortin (POMC). This precursor not only contains the sequence of ACTH, but also the sequences of alpha-melanotropin, beta-melanotropin and the endogenous opiod peptide, beta-endorphin. POMC is synthesized in two regions of the pituitary as well as in distinct sets of neurons in the central nervous system. Although this biosynthetic pathway is present in all extant vertebrates, few studies have attempted to explain how this biosynthetic pathway evoluted. Insights into this issue can be obtained from studies on species that appear to exhibit traits that can be considered ancestral. This study will focus on two "mainline species," the holostean fish, Amia calva, and the Australian lungfish, Neoceratodus forsteri. These species occupy pivotal positions in the phylogeny of the bony fish. The holostean fish are on a line of evolution which gave rise to the teleosts, the modern bony fish, and the lungfush are on the line of evolution which gave rise to the tetrapods. The projects in this proposal will focus on the biochemistry of the POMC end products in these species. These studies will provide new information on the evolution of the pro-opiomelanocortin biosynthetic pathway and will further our understanding of the mechanisms involved in production of chemical signals by endocrine cells.

The biosynthesis of low molecular weight polypeptide hormones is dependent upon a complex sequence of posttranslational processing events. The processing enzymes that mediate these events are found within distinct subcompartments of the ER/golgi/secretory granule network. Understanding the expression, regulation and intracellular routing of these processing enzymes within organelles is central to establishing a clear understanding of the cell biology of secretory cells. Studies on non-mammalian endocrine tissues reveal the array of strategies that have evolved to mediate the biosynthesis of polypeptide hormones. Amphibian model systems will be used to analyzed two aspects of the Proopiomelanocortin (POMC) biosynthetic pathway that are relevant to polypeptide biosynthetic pathways in general: the segregation of processing events within cells, and the developmental regulation of processing enzymes. The toad, Bufo marinus, provides a unique model system to study the N- acetylation of the POMC products, alpha-MSH and beta-endorphin. In mammals, these reactions are performed in parallel in secretory granules. In the intermediate pituitary of anuran amphibians, these reactions are separated both temporally and spatially. A series of studies involving pulse/chase paradigms, immunoelectron microscopy, subcellular fractionation and kinetics analyses will attempt to explain how this amphibian has succeeded in segregating the N-acetylation of these POMC end products. These projects will provide insights into the strategies for routing and regulating processing enzymes within vesicles. Studies on the anterior pituitary of larval Ambystoma tigrinum and neotenic Ambystoma mexicanum will investigate developmental changes in the proteolytic cleavage of ACTH. The developmental shift in ACTH processing observed in amphibian appears to be a common feature of tetrapod corticotrope ontogeny. The amphibian pituitary studies provide convenient models for studying ACTH posttranslational mechanisms, and circumvent many of the limitations of the fetal rat pituitary model system. The ACTH- related cleavage products generated by larval corticotropes will be characterized, and the effects of these products on steroid synthesis by larval adrenal cells will be determined.

IBN-9412115 Robert Dores The pituitary gland is the site for synthesis of several polypeptide hormones which are used as chemical messenger to influence a variety of functions such as growth, reproduction, and chronic stress regulation. One characteristic of polypeptide hormones is that the molecule that is first synthesized inside the cell (called a prohormone) is biologically inactive and must undergo a variety of processing steps by enzymes to convert the non-functional prohormone into a biologically active hormone. One of these important prohormones is proopiomelanocortin (POMC) found in the intermediate pituitary. POMC is the common prohormone for adrenocorticotropin, melanocyte-stimulating hormone, and the endogenous opioid, beta-endorphin. After POMC is synthesized, it undergoes a number of processing steps to make these active hormones. The sites inside the cell where the enzymatic reactions take place and the regulation and nature of the enzymes involved are poorly understood. This is a proposal to study in melanotropic cells of the anuran amphibian, Bufo marinus, the subcellular coordination of two important enzymes (prohormone convertase 1 and 2) that are involved in the processing and the location inside the cell where this processing occurs. Since the proper synthesis of pituitary hormones is essential to the survival of all vertebrates, these studies are critical for providing new insights into the cell biology of pituitary cells.

IBN-9507171 Dores, Robert M. Previous attempts at evaluating phylogeny of vertebrates by comparing sequences of low molecular weight polypeptide hormone families have yielded, in general, unsatisfactory phylogentic trees. This is due to the small size and high degree of conservation, such that there are not adequate differences in peptide structure to construct appropriate trees. Moreover, construction of trees based on these data have lead to errors in establishing the relationship of one species to another. However, Dr. Robert Dores believes that phylogenetically relevant information may be obtained by investigating the structure of the precursor molecules for the pituitary hormones, due to their larger size as opposed to the hormones themselves. In the present studies, Dr. Dores proposes to isolate and sequence the gene encoding proopiomelanocortin from the pituitary of a number of different species. By comparing the sequences of these genes using maximum parsimony procedures, development of an accurate phylogenetic map relating these species with other vertebrates will be possible. This new information about proopiomelanocortin, the precursor molecule for corticotropin, melanocortin and beta-endorphin, a potent endogenous analgesic molecule, will provide more direct evidence for the relationship between several species. In addition, by sequencing the gene encoding proopiomelanocortin, the precursor for the important mammalian stress molecule, corticotropin, new information about stress responses in humans may be obtained.

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.

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.

Title:Co-evolution of the Opioid/Orphanin Gene Family and Cognate Receptor Gene Families.
PI: Robert M. Dores. Institution : University of Denver

Communication among organ systems is accomplished by two intercellular communication systems, the nervous system and the endocrine system, that facilitates the flow of information throughout the body. In these communication networks, critical messages are carried by chemical signals (ligands) that interact in a highly selective manner with specific receptors on a target cell, a "lock and key" strategy. The origin of these ligand/receptor interactions are unknown, but the studies in this proposal will examine species from old lineages in order to reconstruct the transformations that have occurred in ligand-coding and receptor-coding genes during the evolution of distinct chemical communication networks. In order to study the co-evolution of ligand-coding genes and their corresponding receptor-coding genes this proposal will use the opioid/orphanin ligand-coding gene family (the source of opioid and melanocortin ligands), the opioid receptor-coding gene family and the melanocortin receptor gene family, which provide excellent models. The opioid/orphanin ligands influence analgesia, cardiovascular function, and some actions of the immune system, while the melanocortins are involved in chronic stress regulation, fat and glucose metabolism, and feeding behavior. Students will analyze species at critical branch points in the radiation of vertebrates such as: the lungfish, Neoceratodus forsteri, the white sturgeon, Acipenser transmontanus, the horn shark, Heterodontus francisci , the ratfish, Hydrolagus colliei, and the jawless vertebrates, Myxine glutinosa (hagfish) and Petromyzon marinus (lamprey). Studies will integrate the cloning of ligand coding genes with the cloning of their respective cognate receptor-coding genes in these species. The cloned receptors will be expressed in cell lines for binding study analysis, and the distribution of these receptors in the central nervous system and in peripheral tissues will be determined. These studies will reveal novel sequences for both ligands and receptors in the species that have been selected for these projects. Receptor sequence and ligand sequence databases will be created to identify amino acid motifs that could be modified in future site directed mutagenesis experiments to address structure/function related questions. These studies may lead to the development of analogs to the ligands (opioids and melanocortins) that may have therapeutic applications. Projects will be conducted by Ph.D. and MS graduate students and undergraduate honors students. Minority students will be recruited for summer REU (NSF Research Experience for Undergraduates) positions. Finally, a molecular cloning project related to this set of studies will be incorporated into the lab techniques course, BIOL 3655 "Molecular Neuroendocrinology" annually. This proposal fits into the area of Biological Systems Informatics in which questions of genome evolution can be addressed using genomic sequence database analyses. This proposal will both add information to a large ligand/receptor sequence database, as well as "data-mine" the database for new insights into receptor/ligand protein evolution and linked gene co-evolution.