William L. Pak - US grants

The long-term goal of this project is to elucidate the molecular mechanisms of photoreceptor excitation and degeneration using single-step Drosophila mutants isolated for this purpose. Because evidence uncovered during the current project period suggests that the mechanisms of photoreceptor excitation and degeneration are closely related, mutations affecting both processes will be investigated. These mutations define at least 25 genes. Double mutants will be constructed in pairwise combinations to identify a subfamily of interdependent genes among the 25 genes being tested. Detailed genetic, electrophysiological, and morphological analyses will be carried out on the mutants identified by the double mutant studies. Attempts will be made to clone at least one, and hopefully as many as three, genes during the proposed project period. One of the main objectives of isolating these genes is to probe the human genome for homologous sequences. This may be one of the very few viable approaches for isolating human genes involved in photoreceptor degeneration presently available. In addition, additional mutations will be isolated in the ninaE gene, the presumptive R1-6 opsin structural gene, and these mutations will be investigated in detail to carry out structure-function analyses on the gene at the molecular level.

A large number of mutations have been isolated which affect the phototransduction process in D. melanogaster. Such mutants are of importance in understanding certain clinical conditions. Many disorders of the human eye are genetic in orgin, and animal models are extremely valuable to inquire into the genetic bases of these disorders. Drosophila mutants exists which exhibit phenotypes analogous to those observed in human retinal degenerative diseases, such as retinitis pigmentosa. Conditions found in certain forms of color blindness (protanopia and deuteropia) are also observed in several types of Drosophila photoreceptor mutants. The initial objective of the proposed study is to isolate cloned segments of Drosophila DNA from two gene loci known to be involved in photoreceptor function (the norp A and nina E loci). The molecular organization of these genes will be examined in wild type genomic DNA, in addition to DNAs isolated from a large number of different nina E and norp A mutants. In this way it should be possible to determine how changes in the organization and structure of these genes is correlated with photoreceptor potential defects, altered behavioral response, and blindness. The complex complementation pattern observed for nina E mutations will be examined at the molecular level to ascertain whether or not the nina E region contains several distinct but related genes involved in phototransduction. The long term experimental plan also includes the subsequent isolation and characterization of nina E and norp A RNA transcripts and protein products. D. melanogaster DNA sequences exist which appear to be homologous to several cDNA clones prepared from bovine retina mRNA. These bovine homologous regions of the Drosophila genome will be isolated to study their possible genetic role in retina structure and/or function.

The principal participants of the proposed center will consist of the four vision researchers in the Department of Biological Sciences. In addition, we have listed below the names and research interests of the "supporting participants." These are the colleagues who have had various forms of interactions (discussions, consultations, and collaborations) with the principal participants in the past and who are likely to enter more extensive collaborations in the future. At the very least, they help form the intellectual background for the proposed Center. All are from the Department of Biological Sciences (in the School of Science) with the exception of Drs. Kent and Wessermen who are members of the Departments of Biochemistry (School of Agriculture) and Psychological Sciences (School of Humanities, Social Science, and Education), respectively.

A large number of mutations have been isolated which affect the phototransduction process in D. melanogaster. Such mutants are of importance in understanding certain clinical conditions. Many disorders of the human eye are genetic in orgin, and animal models are extremely valuable to inquire into the genetic bases of these disorders. Drosophila mutants exists which exhibit phenotypes analogous to those observed in human retinal degenerative diseases, such as retinitis pigmentosa. Conditions found in certain forms of color blindness (protanopia and deuteropia) are also observed in several types of Drosophila photoreceptor mutants. The initial objective of the proposed study is to isolate cloned segments of Drosophila DNA from two gene loci known to be involved in photoreceptor function (the norp A and nina E loci). The molecular organization of these genes will be examined in wild type genomic DNA, in addition to DNAs isolated from a large number of different nina E and norp A mutants. In this way it should be possible to determine how changes in the organization and structure of these genes is correlated with photoreceptor potential defects, altered behavioral response, and blindness. The complex complementation pattern observed for nina E mutations will be examined at the molecular level to ascertain whether or not the nina E region contains several distinct but related genes involved in phototransduction. The long term experimental plan also includes the subsequent isolation and characterization of nina E and norp A RNA transcripts and protein products. D. melanogaster DNA sequences exist which appear to be homologous to several cDNA clones prepared from bovine retina mRNA. These bovine homologous regions of the Drosophila genome will be isolated to study their possible genetic role in retina structure and/or function.

The long-term goal of this project is to elucidate the molecular mechanisms of photoreceptor excitation and degeneration using single-step Drosophila mutants isolated for this purpose. Because evidence uncovered during the current project period suggests that the mechanisms of photoreceptor excitation and degeneration are closely related, mutations affecting both processes will be investigated. These mutations define at least 25 genes. Double mutants will be constructed in pairwise combinations to identify a subfamily of interdependent genes among the 25 genes being tested. Detailed genetic, electrophysiological, and morphological analyses will be carried out on the mutants identified by the double mutant studies. Attempts will be made to clone at least one, and hopefully as many as three, genes during the proposed project period. One of the main objectives of isolating these genes is to probe the human genome for homologous sequences. This may be one of the very few viable approaches for isolating human genes involved in photoreceptor degeneration presently available. In addition, additional mutations will be isolated in the ninaE gene, the presumptive R1-6 opsin structural gene, and these mutations will be investigated in detail to carry out structure-function analyses on the gene at the molecular level.

The overall objective of this project is to carry out molecular genetic investigations of the mechanisms of photoreceptor excitation and degeneration using Drosophila mutants, with the eventual goal of building a comprehensive picture of photoreceptor function in this organism. Among the specific experiments proposed are further characterizations of the norpA gene, completing the analysis of the brain-specific Go clone, and attempting to isolate eye-specific G protein clones. The heart of the proposal, however, is the drastically altered strategy for gene cloning which would more fully take advantage of the large number of ERG-defective mutants available in this lab. The new approach calls for mapping as many of the genes corresponding to the ERG-defective mutants as possible and, in parallel with mapping, isolating a pool of eye-specific genomic clones. The eye-specific clones that correspond to the mutant genes are to be identified by comparing chromosomal positions of eye-specific clones with those of mutant genes. The identification is then confirmed by various molecular techniques. In parallel with molecular characterization of the genes isolated by the above means, mutants with defects in the genes will be analyzed in detail. Because the phototransduction pathway in Drosophila is prototypical of one of the most widely utilized signal transduction cascades, the understanding of this pathway is likely to be of importance in understanding signal transduction in general. Moreover, insofar as many human disorders are thought to be due to errors in the components of this pathway, the proposed study is likely to contribute to the knowledge needed for eventual prevention and treatment of these disorders.

The long-term objective of the project is to probe the molecular mechanisms of synaptic transmission using a large number of Drosophila mutants that are defective in the responses of the postsynaptic neurons but not in those of the presynaptic neurons. The more immediate objectives are to complete projects now under way and to carry out systematic phenotypic characterizations of putative synaptic transmission-defective mutants. The projects now under way include (1) raising antisera against histidine decarboxylase peptides to determine the site of histamine synthesis and (2) cloning and characterizing the putative calmodulin and histamine receptor genes. Phenotypic characterization of the existing mutants will be carried out for the purpose of identifying a subset of mutants with interesting or interpretable phenotypes. The goal for this project period is to identify up to three complementation groups of such mutants and concentrate the lab's efforts on those mutants. Several possible cloning strategies are described. It is hoped that the combination of phenotypic analysis of the mutants and molecular analysis of the genes identified by the mutants would provide fresh insights into the mechanisms of synaptic transmission. Although it is now widely acknowledged that Drosophila is one of the premier organisms for molecular studies of biological processes, there have been virtually no previous attempts to systematically exploit the power of this organism in the molecular study of synaptic transmission. Insofar as synapses occupy a central position in all complex neural processes, detailed molecular elucidation of synaptic transmission, as envisioned in this proposal, it critically important in the eventual understanding of brain functions and behavior.

Project Summary/Abstract The overall long-term objective of the research program in this laboratory is to elucidate the mechanisms of photoreceptor excitation by molecular genetic analyses of ERG (electroretinogram)-defective Drosophila mutants. There are two broad specific aims for the coming project period. In one, we will attempt to definitively identify the molecule that acts as the excitatory messenger to light-activated channels in Drosophila photoreceptors, TRP (transient receptor potential) &TRPL (TRP-like). The founding member of the TRP channel superfamily was discovered in Drosophila, and our lab played a prominent role in the early TRP work and coined the term, TRP. TRP channels are now known to play a key role in many human diseases. Understanding the mechanism of TRP activation is important in providing insights into the underlying causes of many of these diseases. In the second specific aim, we propose to investigate the mechanisms of visual defects found in three Drosophila mutants from three different genes, all of which were generated by chemical mutagenesis. A combination of genetic, molecular, cell biological, and electrophysiological approaches will be used for the analyses of these mutants. Two of these mutants have defects in quenching rhodopsin molecules after they are photo-excited. For one of the mutants, the gene that carries the mutation responsible for the mutant phenotype has been identified to be a Drosophila ortholog of a human Usher syndrome gene. Usher syndrome is the most common autosomal disorder of hearing and vision, characterized by congenital hearing loss, vestibular dysfunction, and retinitis pigmentosa. How mutations in Usher syndrome genes cause retinitis pigmentosa is not known. The study of this mutant may lead to the understanding of how a mutation in at least one Usher syndrome gene causes retinitis pigmentosa. The third mutant is impaired in Ca2+-dependent light adaptation. Investigation of this mutant could lead to the identification of a protein involved in the light adaptation process. Loss of light adaptation is a serious visual impairment. Identification of a molecule that plays a critical role in adaptation would be an important contribution to understanding of the adaptation process, and it could lead to the development of therapeutic intervention for this form of visual impairment

DESCRIPTION (provided by applicant): The objective of this project is to clone and characterize up to eight genes identified by Drosophila mutants defective in synaptic transmission between photoreceptors and their target neurons. These mutants have also been shown to display specific impairments of synaptic transmission at the larval neuromuscular junction, indicating that the mutations affect basic mechanisms of synaptic transmission common in multiple classes of synapses. Because these mutants were isolated by chemical mutagenesis, however, traditional approaches to cloning the corresponding genes are tedious and time-consuming. We propose a novel approach to cloning these genes using whole-genome DNA microarrays. A major strength of this application, in addition to the use of the novel approach, is that it involves the collaborations of three investigators with complementing expertise: Dr. William L. Pak, Dr. Kendal Broadie, and Dr. Rebecca Doerge. Dr. Doerge, a statistical geneticist, will collaborate in the planning of microarrary experiments, and in the statistical analysis and interpretation of the microarray data. Dr. Pak's laboratory will have the major responsibility for carrying out the microarray experiments, and Dr. Broadie and Dr. Pak will share in the analysis of mutants. The ultimate goal of the project is to identify proteins important in synaptic transmission and characterize their functions, particularly those proteins which have homologs in mammals but whose functions have not been identified. Insofar as synapses occupy a central position in all complex neural processes, elucidation of synaptic processes are critically important in the understanding of brain functions and behavior, and hence in the understanding and management of mental illnesses. Although this work will be carried out using Drosophila because of its experimental tractability, the work is expected to lead to the identification of novel mammalian homologs and insights into their functions.