John Carlson - US grants

The organization and development of neuronal systems are problems of great importance in modern biology. This proposal concerns a molecular and genetic analysis of a chemosensory system and is designed with a view to addressing the fundamental problems of (i) the mechanisms of chemosensory transduction, (ii) the principles of chemosensory coding, and (iii) the means by which genes specify the development of a complex neuronal system. Drosophila is an excellent organism in which to address these problems, as it possesses a highly sophisticated olfactory system which may be investigated by powerful genetic and molecular means. Among a set of mutants defective in chemosensory behavior are several which respond abnormally to one chemical but which appear normal in response to other chemicals. Such mutants define genes which may play roles in chemically-specific olfactory pathways; these genes could encode olfactory receptor molecules or other products required for transduction or processing of specific types of olfactory information. A genetic and molecular analysis of one such gene, defined by the 3D18 mutation, is proposed. By characterizing the distribution within the olfactory system of products of genes which are specific to individual functional pathways, we anticipate that we may gain an enhanced understanding of the functional organization of the system, which is, in turn, critical to an understanding of how complex olfactory stimuli are encoded into a form interpretable to the brain. Genetic analysis of the olfD locus is also proposed; we aim to determine whether olfD represents a second class of gene, required for response to all chemicals but not in other sensory modalities, such as vision. Other mutants we have isolated are defective both in visual physiology and chemosensory behavior. Relationships between chemosensory and visual transduction will be sought by determining whether the affected genes correlate with previously characterized visual system genes, and by subjecting visual mutants to chemosensory assays. The feasibility of using a direct molecular approach to isolate genes required for olfactory transduction and information processing will be assessed: we will attempt to isolate antennal- specific cDNA clones using a new method of subtractive hybridization.

An understanding of the influence of local landscape structure on insect population dynamics is critically needed to address questions concerning agro-ecosytem re-design. An in depth examination of specific alfalfa herbivores and predators in a diverse ecological-agricultural landscape is proposed. A Geographic Information System (GIS) will be employed as the principal analytical tool in establishing relationships between landscape complexity and insects. The following objectives are proposed: characterize local ecosytems wide distribution of predaceous coccinellids, aphid prey and leafhopper migrants as colonization of alfalfa begins; quantify microclimate factors which influence insect development in and dispersal from over- wintering sites to spring habitats; measure and determine the effect of landscape structural diversity on initial colonization of alfalfa by insect predators and pests, and the recolonization of alfalfa following disturbance associated with alfalfa cutting and harvest. These results will be used in a GIS assessment of landscape effects on insect dynamics. This research will provide essential information on predator/prey dynamics across landscapes.

This award to the Michigan Meteorological Resources Program (MMRP) at Michigan State University will enhance instruction and research in the areas of meteorology and climatology. Funds are provided for the acquisition of an interactive meteorological computer system (UNIDATA/PC- McIDAS). This computer system will provide hardware and software for the near-real-time display of satellite images and conventional and diagnostic parameters. Contributions of PC-McIDAS to instruction include providing students with experience in interpreting satellite imagery and permitting classroom and laboratory demonstrations of the relationships between upper-air features, surface patterns, and cloud systems. Satellite images also will be archived for research purposes. In particular, this archive will be used for further research concerning the climate of Michigan.

Post-traumatic stress disorder (PTSD) is a serious, complex anxiety disorder that may result from exposure to significant natural and social trauma, including war. Approximately 500,000 (15%) or more of Vietnam veterans in the United States are afflicted with this disorder presenting an immediate and long term health problem of major proportions that impacts on nurses, psychologists, and other health professionals. The long-term objectives of this proposal are: a) to provide the most effective forms of a care and diagnosis for combat-related PTSD in the multi-ethnic population of Vietnam veterans utilizing the Veterans Administration (VA) Medical Center in Hawaii; b) to promote research on treatment and assessment alternatives for stress-related disorders, especially emphasizing "multi-modal" approaches, and c) to enhance research and training opportunities and resources for students and faculty in the School of Nursing and Health Psychology programs at the University of Hawaii. These objectives will be addressed through collaborative research between the School of Nursing, Department of Psychology, and VA Medical Center that aims specifically to: 1) assess PTSD in a population of Vietnam veterans representing Pacific Islanders, Asian-Americans, and Caucasians; b) employ diagnostic instruments that target physiological, behavioral, and cognitive modes of PTSD; and c) perform controlled clinical research that compares biofeedback-assisted relaxation training and 'imaginal flooding' methods with treatments that emphasize routine clinical care and control for attentional effects. In each experiment, male Vietnam veterans with a diagnosis of PTSD will be assigned to treatment and control conditions in equal numbers to cells representing the three ethnic subgroups. Following preliminary and baseline assessment, in the first experiment, a general relaxation intervention including multi-muscle biofeedback training will be applied. In a second experiment, an imaginal-flooding intervention will be applied that emphasizes the repeated rehearsal of trauma-laden memories and attendant emotional arousal. In both studies, the treatment effects will be assessed using physiological and questionnaire-interview instruments. In addition, the experimental design allows for treatment to be provided for the control subjects, ensuring equitable treatment for all subjects and optimum utilization of the subject population for treatment and multi- ethnic comparisons. The results of this research will have implications for behavioral (associative learning) and psychophysiological theories of stress-related disorders, and will foster the development of future treatment and assessment strategies for this serious disorder.

The organization and development of neuronal systems are problems of great importance in modern biology. This proposal concerns a molecular and genetic analysis of a chemosensory system and is designed with a view to addressing the fundamental problems of (i) the mechanisms of chemosensory transduction, (ii) the principles of chemosensory coding, and (iii) the means by which genes specify the development of a complex neuronal system. Drosophila is an excellent organism in which to address these problems, as it possesses a highly sophisticated olfactory system which may be investigated by powerful genetic and molecular means. Among a set of mutants defective in chemosensory behavior are several which respond abnormally to one chemical but which appear normal in response to other chemicals. Such mutants define genes which may play roles in chemically-specific olfactory pathways; these genes could encode olfactory receptor molecules or other products required for transduction or processing of specific types of olfactory information. A genetic and molecular analysis of one such gene, defined by the 3D18 mutation, is proposed. By characterizing the distribution within the olfactory system of products of genes which are specific to individual functional pathways, we anticipate that we may gain an enhanced understanding of the functional organization of the system, which is, in turn, critical to an understanding of how complex olfactory stimuli are encoded into a form interpretable to the brain. Genetic analysis of the olfD locus is also proposed; we aim to determine whether olfD represents a second class of gene, required for response to all chemicals but not in other sensory modalities, such as vision. Other mutants we have isolated are defective both in visual physiology and chemosensory behavior. Relationships between chemosensory and visual transduction will be sought by determining whether the affected genes correlate with previously characterized visual system genes, and by subjecting visual mutants to chemosensory assays. The feasibility of using a direct molecular approach to isolate genes required for olfactory transduction and information processing will be assessed: we will attempt to isolate antennal- specific cDNA clones using a new method of subtractive hybridization.

This is a proposal for funds to purchase a research-grade light microscope for four projects: (i) a molecular genetic analysis of the Drosophila olfactory system; (ii) a molecular and developmental analysis of the Drosophila E75 gene, a model system for analysis of steroid-triggered regulatory hierarchies; (iii) a molecular, genetic, and developmental analysis of the positional control of leaf cell differentiation in maize; (iv) an analysis of the developmental regulation and function of the mammalian transcription factor AP-2. For each of these projects, a high-quality light microscope facility is crucial.

Recent work has shown major similarities across a wide range of organisms in the biochemical mechanisms for olfactory reception. The fruit fly, Drosophila, provides an excellent model system for study because of its accessibility for genetics, molecular analysis, behavior, and even some physiology. This project combines these techniques to address the molecular basis for olfactory sensitivity. Modern genetic technology and methodology is used to isolate particular mutant strains of Drosophila that show particular olfactory sensitivity or loss. These mutants allow identification of particular genes by the pattern of proteins that are expressed. Genes that are expressed specifically in olfactory tissue during development will be characterized, and their expression or deletion correlated to specific kinds of olfactory behavior. One of these genes apparently is expressed differently in males and females, suggesting an important role in mate recognition or other important reproductive behavior. Results will be important for understanding the mechanisms of olfactory reception at the fundamental level of molecular genetics, and will have a broader impact as well on sensory neuroscience and on genetics, with potential applications including pest control.

DESCRIPTION (provided by applicant): This proposal aims to reveal basic principles underlying olfactory system function, organization, and development. The experimental plan takes advantage of the fruit fly Drosophila as a model system, which allows powerful genetic analysis and convenient physiological measurement of individual olfactory receptor neurons. The project seeks to define and characterize the diversity of olfactory receptor neurons that underlie odor coding in Drosophila, and to understand the molecular mechanisms that generate this diversity during development. A systematic analysis of the receptor neurons of the antenna will be continued in order to define the cellular basis of odor coding. The odor-response spectra and response dynamics of these neurons will be characterized in detail. The acj6 POU domain transcription factor will be analyzed to determine the molecular mechanisms by which it acts in establishing receptor neuron identity. Special attention will be accorded to its role in the regulation of odor receptor gene expression. Little is known about the means by which individual olfactory receptor neurons select, from among a large repertoire, which receptor genes to express. This project aims to identify components, both cis-acting and trans-acting, that are required for the process of receptor gene choice. One goal is to test the hypothesis that receptor gene choice is made in part through a combinatorial code of POU and LIM domain transcription factors. Hundreds of millions of people are afflicted by diseases carried by insects, many of which recognize and locate their human hosts largely through olfactory cues. Advances in the understanding of insect olfaction could lead to new means of controlling these insect pests.

This multidisciplinary investigation, designed specifically for collaborative research among undergraduates, will employ a range of experimental approaches to determine how behavioral decisions of food-hoarding animals influence the dispersal, establishment, survival, eco-physiology, and genetics of oaks over a broad geographic area. Previous research by two of the PI's indicates that caching decisions of mammals results in the selective consumption of acorns of the white oak group (WO) and dispersal, hoarding, and frequent survival of those of the RO group (RO). This study will rely on five approaches to examine the broad evidence for, and implications of, this differential dispersal hypothesis. These are 1. behavioral experiments to further examine patterns of oak dispersal; 2. seed dispersal experiments to determine factors controlling seedling establishment; 3. a broad-scale biogeographic comparison of seedling distributions; 4. an eco-physiological comparison of RO and WO seedlings in the field and lab; and 5. a molecular (PCR) analysis to document comparative genetics and seedling shadows of oak. The collaboration will bring together 15-20 undergraduates per year from biology, chemistry and environmental sciences from two undergraduate institutions. The study will employ a team approach to research (across universities) and a vertical training/learning environment in order to maximize student experience in all aspects of the scientific process, including presentations at conferences and publications. The program will include development of a new course in plant-animal interactions and an annual project conference aimed at promoting student learning.

DESCRIPTION (provided by applicant): The long-term goal of this project is to reveal the logic by which tastants are encoded, and to elucidate basic principles of the organization and development of the neurons that encode them. The experimental plan takes advantage of the fruit fly Drosophila melanogaster as a model system, which allows incisive molecular genetic analysis of taste genes as well as physiological analysis of taste function. The first aim is to complete a functional analysis of a numerically simple model taste organ, the foreleg. The taste neurons of this organ have been defined and all members of the Gr family of taste receptors have been mapped to them. Physiological responses of this organ to sugars, bitter compounds and amino acids will be analyzed with a view to understanding the role of this organ in the evaluation of taste. The analysis is designed to address the problem of how a sensory system integrates the multiple inputs that are ultimately translated into a behavioral response. The results may support a model explaining how the animal makes a decision critical to all animals: whether to accept or reject a potential food source. The underlying basis of feeding regulation has major implications for public health. The second aim addresses the role of G proteins in taste neuron signaling and development. The role of these proteins in chemosensory signaling is a central question in the field. The analysis will test the hypothesis that complete removal of certain G proteins leads to a complete loss of physiological response to either sugars or bitter compounds. The hypothesis that these proteins act in the development of taste neurons will also be tested. The third aim is to examine the function of a bitter receptor by expressing it in cells that have no bitter response. The analysis is designed to test the hypothesis the certain members of the Gr family act as co-receptors for other members. It is also designed to determine whether an efficient system can be constructed for the study of bitter receptors and for the identification of tastants that activate or inhibit them. The results could yield a wealth of new opportunities to study the function of bitter receptors and their role in the perception of bitter taste. Diseases carried by insects afflict hundreds of millions of people each year, and these insects receive taste cues from their human hosts. Advances in the understanding of taste may lead to new means of controlling these insect vectors of human disease. In particular, the identification of compounds that activate or inhibit bitter taste receptors could provide new agents for the control of insect vectors and the diseases they transmit.

DESCRIPTION (From the Applicant's Abstract): This project is designed to reveal basic principles of olfactory system function and development, through a detailed analysis of the recently discovered DOR family of odorant receptor genes. The proposal addresses fundamental issues of receptor function, taking advantage of the strengths of Drosophila as an experimental system. Olfaction is an ancient sensory modality, and many principles of olfactory function and organization are well conserved among invertebrates and vertebrates. Moreover, understanding of insect olfaction may be useful in controlling insect vectors of human disease that find their human hosts or their mates through olfactory cues. The proposal aims first to identify and classify all members of the DOR gene family, including highly divergent members. Extant mutations affecting DOR genes will be sought. The distribution of receptors in the mature olfactory system will be addressed in two ways. First, the recent isolation of an antibody against a DOR protein allows a high-resolution analysis of receptor localization in the olfactory system. Results showing that the DOR22A.2 protein is present in the cavities of olfactory sensilla, as expected of an odorant receptor localized to dendrites, will be confirmed and extended by immunoelectron microscopy and with antibodies against additional DOR proteins. Second, the number of receptors per neuron will be investigated by double-label in situ hybridization. A developmental analysis of receptor expression is proposed. The developmental profiles of all DOR genes will be analyzed, with a view to determining how many genes are expressed early in development, as is DOR22A.2. Preliminary immunohistochemical results indicating that the DOR22A.2 receptor is present on the axons of olfactory receptor neurons will be confirmed and extended. A genetic analysis of selected DOR genes is proposed to test the hypothesis that they encode odorant receptors. DOR gene function will be altered through overexpression and by loss-of-function mutations. These experiments may identify a ligand for a DOR protein. They may also provide functional evidence regarding the number of receptors expressed per neurons, and the possibility of developmental roles for receptors.

0207202
de Pamphillis

This research project is designed to accomplish two aims: Aim 1: To construct and array high-quality BAC libraries to provide a genomic resource on a wide range of species. Aim 2: To enable researchers working with green algae, non-seed land plants, and seed plants (including flowering plants) to identify genes critical for understanding plant form and function and how land plants arose and diversified. This project will enable progress toward an understanding of the genetic basis for the transitions that mark the most fundamentally important steps in green plant evolution. The Deep Green community (http://ucjeps.herb.berkeley.edu/bryolab/deepgene/index.html) will help to provide an infrastructure for ongoing scientific exchange. Bioinformatics and a web site will be provided to the community to access these resources (http://www.genome.clemson.edu/).

Selected Species: Desired coverage is given in parentheses. Where two coverage values are listed, two libraries using different restriction enzymes will be made.

Green algae:
Volvox carteri (5x, 5x)
Caulerpa mexicana (8x)
Mesostigma viride (8x)
Coleochaete orbicularus (6.4x, 6.4x)
Chara aspera (6x)

Non-seed plants:
Marchantia polymorpha (8x)
Anthoceros sp. (6.3x, 6.3x)
Lycopodium lucidulum (5.1x, 5.1x)
Angiopteris erecta (6.2x, 6.2x)
Ceratopteris richardii (4.8x, 4.8x)
Marsilea quadrifolia (5.6x, 5.6x)

Seed plants:
Amborella trichopoda (5.5x)
Nuphar adventa (7x)
Acorus gramineus (7x)
Lirodendron tulipifera (7x)
Mimulus guttatus (7x)

DESCRIPTION (provided by applicant): Continuing support is requested for Yale's Predoctoral Training Program in Genetics. The program involves 66 trainers from a total of 11 different departments. The principal administrative and training entities are the Departments of Molecular, Cellular and Developmental Biology (MCDB) and Ecology and Evolutionary Biology (EEB) in the Yale University Faculty of Arts and Sciences, and the Department of Genetics in the Yale University School of Medicine. The numbers of graduate students currently enrolled in the primary participating departments are 66 (MCDB), 54 (Genetics) and 24 (EEB). Students in the Departments of MCDB and Genetics enter the university through the interdepartmental Combined Program in the Biological and Biomedical Sciences. Most incoming trainees have a Bachelor's degree in biology or biochemistry. Admission is granted to trainees whose graduate record exam scores and grade point averages are high. Particular attention is paid to research accomplishments and letters of recommendation from scientists with whom the trainee has done research. The program offers training in all aspects of modem genetics, including clinical genetics, genomics and bioinformatics, developmental genetics, immunogenetics, cancer genetics, evolutionary and population genetics. Students have the opportunity to work with a variety of model organisms, including Drosophila, C. elegans, yeast, Arabidopsis, maize, E. coli, viruses and mice. Training in the first year includes formal course work and research rotations. In the second year, students continue course work and begin thesis research. As part of the qualifying exam administered in year two, each student is expected to demonstrate mastery in a number of broad topic areas and to prepare and defend one or more research proposals. Later years are devoted to thesis research and preparation of a dissertation. All students receive supervised teaching experience. The majority of graduates go on to do postdoctoral research, and many of these subsequently obtain independent positions at academic and research institutions. In addition, many graduates obtain research positions in biotechnology and pharmaceutical companies.

Intellectual Merit: Rapid advances in technology are fundamentally altering the biological sciences. Once relegated to extremely expensive and ponderously slow "Big Science" projects, the sequencing, assembly, and annotation of large genomes, such as those found in plants, is increasingly becoming the province of smaller, less expensive consortia. Full inventories of expressed genes can now be obtained for any organism at very modest cost. This "genomics revolution" is touching all aspects of biology, not the least, the study of plant biology. With the increasing ease of obtaining genomic data, the focus must now turn to producing information from the data and synthesizing approaches from sub-disciplines which have historically operated separately. In particular, a detailed understanding of genome evolution, not just individual genes, is becoming an attainable goal. Correlating these molecular data with physiological responses, ecosystem interactions, and crop productivity is a major scientific goal. Towards this end, the 18th Penn State Symposium in Plant Biology: Plant Evolutionary Genetics and Genomics will be held May 18-21, 2011 at Penn State's University Park campus. This meeting will bring together both leading scientists and early-career researchers to exchange results and develop collaborations in this rapidly evolving field. The symposium is the 18th in a very successful series of Plant Physiology / Plant Biology Symposia, which have been held at Penn State since 1986.

Broader Impacts: This meeting will provide critical educational opportunities to undergraduate students, graduate students, post-doctoral researchers, and to early-career faculty members in the following ways: 1) Several speakers will be invited to deliver short talks based upon the quality of submitted poster abstracts. These decisions will emphasize inclusion of early-career faculty members and members of groups under-represented in American science. 2) Travel awards to offset costs of attendance to the meeting will be distributed to select attendees, based upon demonstrated need and upon other factors, with a preference towards undergraduates at small, non-research intensive colleges/universities. 3) Ample time in the symposium schedule will be "unstructured" time at poster sessions and in proximity to refreshments. This arrangement allows maximum interaction between students and the cadre of distinguished scientists serving as plenary speakers. In addition, it is important to note that the topic of our symposium is in a critical area of current science: Increased understanding of plant genetics and genomics may play a key role in the development of a sustainable biofuels industry in the United States and world-wide.

PI: John E. Carlson (Pennsylvania State University)

Co-PIs: Jeanne Romero-Severson (University of Notre Dame), Scott E. Schlarbaum (University of Tennessee - Knoxville), Mark V. Coggeshall (University of Missouri - Columbia), Haiying Liang (Clemson University), Oliver Gailing (Michigan Technological University), and Ketia L. Shumaker (University of West Alabama).

Senior Personnel: Meg Staton (Clemson University) and Nicholas C. Wheeler (Oregon State University).

Most timberlands in the United States are natural forests, of which eastern hardwood forests comprise more than half. The eastern hardwood forests are complex biological systems, covering over 400 million acres of bottomland and riparian sites, major watersheds, mesic sites and upland xeric sites. These forests provide habitat and food for wildlife, stabilization of riparian zones, long-term carbon sequestration and other essential ecosystem services as well as wood and biomass products for human use. The increasing incidence of introduced exotic pests, diseases and invasive plants, combined with climate change and forest fragmentation, threaten the sustainability of these forest ecosystems. Unfortunately, few genomic resources are available for use in studying the consortium of hardwood species that compose the eastern forests. An interdisciplinary team will work together to develop new genomic resources for important species that represent the major taxonomic groups of eastern hardwood trees, from the oldest to more recently evolved, including yellow poplar (Liriodendron tulipifera), sweetgum (Liquidambar styraciflua), honey locust (Gleditsia triacanthos), northern red oak (Quercus rubra), black walnut (Juglans nigra), sugar maple (Acer saccharum), blackgum (Nyssa sylvatica), and green ash (Fraxinus pennsylvannica). The project will produce sequence databases for expressed genes, genetic markers, genetic linkage maps, and reference populations This will provide lasting genomic and biological resources for the discovery and conservation of genes in hardwood trees for growth, adaptation and responses to environmental stresses such as drought, heat, insect pests and disease. These resources will be available to the scientific community and the public through the project website (www.hardwoodgenomics.org). All original sequence data will be deposited in NCBI's Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra) and the genetic linkage maps and associated marker data will also be available at the Dendrome database (http://dendrome.ucdavis.edu/).

The broader impacts from this project will include forest health, tree improvement, forest management, molecular evolution, scientific training, and public education. An increasing incidence of exotic pests and diseases, combined with climate change and forest fragmentation, are threats to the sustainability of forest ecosystems and economies. This project will provide powerful new tools to address such forest health issues and the protection and restoration of forest genetic diversity and productivity. The project will also fill gaps in available genomic resources for important groups of flowering plants, including the taxonomic orders Magnoliales (yellow poplar), Proteales (sweetgum), Fabales (honey locust), Fagales (Northern red oak and black walnut), Sapindales (sugar maple), Cornales (blackgum), and Lamiales (green ash). These resources will enrich the scientific community's ability to study the evolution of not only woody plants, but also all angiosperms at a resolution and depth not previously possible. All of the data generated by the project will be deposited in high-visibility public community databases, and all gene clones, libraries, and reference population DNAs will be stored and available to the public at cost. Descriptions of the resources and analyses of the results will also be published as journal articles, at national and international meetings, and through a public web portal hosted by the Clemson University Genomics Institute. This project will also provide for the training of undergraduate, graduate and postdoctoral students in comparative genomics, evolutionary genomics, population genetics, bioinformatics and forest genetics. Educational programs on plant genomics will be developed for Native American public schools in cooperation with the Cherokee Nation, and substantive research experiences will be provided to minority undergraduate students in collaboration with the University of West Alabama.

? DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate basic principles of chemosensory perception. It seeks to explain at the molecular and cellular level how chemosensory information is encoded. The experimental plan takes advantage of the fruit fly Drosophila melanogaster as a model system, which allows incisive molecular genetic analysis of chemosensory receptors and neurons, and of the functions that they perform. The project focuses on a family of 30 predicted chemosensory receptors, the IR20a clade, that are expressed in chemosensory neurons. The project considers a kind of chemical information that underlies one of the most ancient and fundamental of biological problems: how an animal recognizes a suitable mate of its own species. An understanding of the molecular and cellular basis of species recognition could lead to new means of controlling insects that transmit disease to humans. The first aim examines two ionotropic receptors that are expressed in male neurons that are activated in a species-specific manner. The receptors in one species will be genetically replaced by their counterparts from another. The effects of this switch will be analyzed to test hypotheses about the molecular basis of species recognition. The second aim considers another ionotropic receptor that may act in females as a detector of male cues. The role of this receptor and the neurons in which it is expressed will be analyzed in detail. The results may support a model in which these neurons act in a checkpoint: if they receive an appropriate cue, an acceptance signal is sent to the central nervous system. The third aim capitalizes on an opportunity to analyze the role of a greatly understudied chemosensory organ, the wing. Different neurons on the wing are activated by different chemosensory cues, and an ionotropic receptor has been found to be expressed in a subset of these chemosensory neurons. The role of this receptor and the neurons in which it is expressed will be examined to determine if they are sensitive to species-specific cues. The results could provide a major advance in our understanding of a chemosensory organ whose function has remained speculative for 35 years.

PROJECT SUMMARY The long-term goal of this project is to reveal the molecular logic by which bitter tastants are detected and encoded. The experimental plan takes advantage of the fruit fly Drosophila as a model system, which allows incisive molecular genetic analysis of taste genes and physiological analysis of taste function. The project focuses on a large family of Gustatory receptors (Grs), many of which mediate responses to bitter compounds. The project examines the bitter-sensitive neuron that expresses the fewest Grs in the major taste organ of the head. The first aim will systematically test the functional necessity of each of the Grs expressed in this neuron. This aim is designed to test a model in which a network of coexpressed Grs interact with each other both positively and negatively. The analysis will test the hypothesis that some Grs are ?tuning Grs? that bind tastants and that other Grs are coreceptors. The second aim will systematically test the functional sufficiency of each Gr in the neuron. Using CRISPR technology we will construct an ?empty bitter neuron? that expresses no Grs. We will determine whether individual Grs expressed alone in a bitter neuron are sufficient to confer taste response. This aim could establish a useful in vivo expression system for taste receptors. The third aim will systematically examine a receptor in combination with others to identify partners with which it interacts functionally. The aim will test the hypothesis that there is a combinatorial logic to taste detection, with different combinations of Grs responding to different tastants. This combinatorial logic could enhance the ability of a small number of receptors to detect a large number of tastants. Diseases carried by insects afflict hundreds of millions of people each year. These insects detect their human hosts, their food, or their mates largely through their chemosensory systems. Advances in understanding these chemosensory systems may lead to new means of manipulating them and of thereby controlling insect vectors of human disease.