Seymour Benzer - US grants

9408718 Abstract This project is a continued exploration of the use of Drosophila in genetic approaches to the nervous system. The analysis is applied to problems in development, including the assembly of the eyes and in the formation of their connections to the brain. Special attention is paid to the role of glial cells in these processes, and in their interaction with neurons in maintenance of the integrity of the brain. Genetic analysis is applied to the mechanism of transmitter release at synapses, characterization of the proteins involved, and the effects on behavior of mutations that alter synaptic proteins. In addition, new molecular genetic techniques are described for isolating mutations in selected genes that are expressed in specific parts of the nervous system, and for generating monoclonal antibodies and associated cDNAs for the corresponding gene products. %%% Given the numerous molecular homologies between the fly and other organisms, including mammals, Drosophila serves as a model system for understanding basic mechanisms in the relation of genes to behavior. ***

The proposal is to use molecular genetic methods with Drosophila to tackle problems of differentiation and patterning at various levels, ranging from the establishment of the anterior-posterior axis of the embryo through to the adult with its complex morphology and intricately connected nervous system. The specific subprojects are: "Localization of Pattern Determinants in the Drosophila Egg", "Patterns of Gene Activation in the Drosophila Embryo", "The Bithorax Complex as a Microgenome", "Genetic Specification of Cellular Identity in the Drosophila Nervous System", and "Specification of Neuronal Shape and Connectivity in Drosophila". These five projects utilize very similar methodology, require similar kinds of materials and equipment, and lend themselves to mutual assistance and strong intellectual interaction. The first proposal aims to search for cDNAs representing RNAs localized to either the anterior (A) or posterior (P) poles of the unfertilized egg, with a view to investigating their roles in establishing embryonic pattern, as well as the molecular mechanisms of RNA localization. The second proposal is a molecular genetic and biochemical approach to dissecting the cis-regulatory region required for the expression of the ftz gene in a pattern of stripes along the A-P embryonic axis. The third is to focus on the cis- regulation of the subset of the 400 kb bithorax complex that controls the development of the second through the ninth abdominal segments. This subset includes the homeodomain-containing infra- abdominal genes and additional regions that are required in cis to insure proper development of the abdomen. The fourth proposal is to use specially designed cloning vectors to identify cDNAs that represent rare mRNAs. Preliminary experiments have shown that very large numbers of distinct clones can be obtained and it is proposed to focus on the roles of the corresponding genes in specifying neuronal cell types. The fifth proposal is a genetic and molecular analysis of a gene involved in the formation of a particular identifiable synapse in Drosophila, and the mechanisms by which the genome affects the specificity of neuronal connections.

Humans and flies display a wide variety of genetic defects affecting the eye. Our goal is to elucidate the functions of genes expressed in the human retina by using Drosophila as a model system. We will isolate human genes with homology to Drosophila eye genes, and Drosophila homologs of human genes involved in retinal disease, and use them for functional studies in Drosophila. A set of previously isolated eye-specific Drosophila cDNAs will be used to isolate potential functional human retinal cDNA homologs. The Drosophila cDNAs include several with known sequence homology to human eye genes, such as arrestin and rhodopsin, as well as others of novel sequence. Mutations will be induced in the fly genes corresponding to the Drosophila eye cDNAs, and the resultant phenotypes characterized for anatomical and functional defects in the retina and visual transduction pathways. We have also identified other Drosophila genes based on striking defects in retinal differentiation, or expression patterns of interest to visual system development or function. These genes are currently being cloned in our laboratory, and will also be used to isolate candidate human homologs. To test whether a putative human homolog expresses a similar function in the fly, we win attempt to rescue the fly mutant phenotype with the human cDNA. This will be done by constructing hybrid genes, in which Drosophila promoter sequences are linked to the protein-encoding regions of the human homologs. These constructs will be tested for gene and protein expression in Drosophila tissue culture cells, then introduced into the Drosophila germ line by P element-mediated transformation, and analyzed for expression in the fly. Partial or full rescue of the phenotype will be evidence for conservation of function. This methodology will be tested by using human rhodopsin to rescue the phenotype of the Drosophila rhodopsin mutant, hinae. We will also search for Drosophila homologs of the human retinoblastoma gene, for which a clone is available. Drosophila homologs of the gene will be searched for by low stringency hybridization and PCR amplification of regions of the gene conserved through vertebrate evolution. The function will be investigated by the molecular and genetic methodologies available in Drosophila.

neural degeneration; neurotrophic factors; lethal genes; brain; aging; suppressor mutations; genetic transcription; genetic mapping; membrane proteins; neural plasticity; molecular cloning; transfection; Drosophilidae; laboratory mouse; laboratory rabbit;

Toward the discovery of life assurance genes, we are performing a screen for extended lifespan in Drosophila melanogaster by single gene mutations. The mutant line methuselah consistently displays an approximately 40 percent increase in average lifespan. Interestingly, the mutant flies also show enhanced resistance to conditions of stress, including starvation, low humidity, and high temperature. Upon exposure to paraquat, a free radical generator, they survive longer than control flies. Excision, along with adjacent DNA, results in embryonic lethality in homozygotes, suggesting that the gene also plays an important role during development. The gene structure predicts a protein sequence containing seven hydrophobic regions with homology to various G-protein-coupled, seven-transmembrane-domain cell receptors. This finding suggests that Drosophila can use signal transduction pathways to modulate stress response and aging. This project is to analyze the mechanism involved in methuselah, and to undertake an expanded project for the isolation and analysis of additional life assurance genes.

DESCRIPTION (provided by applicant): Obesity is a complex disorder caused by an imbalance between food intake and energy expenditure. These processes normally are precisely regulated, so that body weight can remain constant over a long time, in spite of variable food intake and activity. This homeostasis suggests the presence of strong regulatory mechanisms, and most biological mechanisms have an underlying genetic basis. The isolation of the Drosophila adipose mutation in the 1960's demonstrated that Drosophila can become obese, since that mutation causes a doubling in the overall fat content of adult flies. The strength of Drosophila as an experimental organism lies not only in its amenability to large scale, fast, and cheap genetic screening, but also in many years of genetic analysis, climaxing with the complete sequence of its genome, which makes it relatively easy to identify and clone genes with interesting mutant phenotypes. The list of biological problems for which Drosophila has been used successfully as a model is impressive, including the identification of genes that regulate the circadian rhythm, aspects of behavior such as learning and memory, and nervous system development. Many of these genes have provided clues to the discovery of their mammalian homologs. The use of Drosophila in obesity research opens the prospect of large-scale genetic screens to identify genes responsible for appetite control, body weight regulation, and fat storage, as well as analysis of the underlying biological mechanisms. We have isolated a series of obese mutants of Drosophila. The specific aims of the project will be to characterize their phenotypes, clone the genes, analyze the affected biochemical pathways, and isolate suppressor genes to reverse the genetic obesity defects.

The survival of an organism depends on its ability to respond appropriately to environmental stimuli. In higher organisms, this response is mediated through the function of the nervous system. To maximize adaptability, the nervous system must not only distinguish between qualitatively different stimuli, but also their intensities. Two major properties allow the nervous system to achieve this goal. First, the nerve impulses produced by a given stimulus travel via a specific neuronal network. Second, the threshold and frequency of the nerve impulses generated by a given stimulus can be modified by external conditions. The information necessary for building both the neuronal network and its components for nervous impulse generation are encoded by the genome of the organism.
One of the major intellectual challenges of modern biology is to understand how this genomic information is translated to an appropriate axonal network, and how those networks function to produce appropriate behavior.
Genetic analysis of behavior is a powerful approach to these problems. It is typically carried out in three stages. First, a paradigm is designed in which the behavior of interest can be easily observed and scored in normal individuals. The second stage uses mutagenesis to identify mutant genes that show clear deviations in behavior. Third, modern genetic techniques make it possible to either shut down or activate specific regions of the nervous system, to identify the circuits within the neural network that produces the behavior.
The purpose of this project is a genetic analysis and silencing of specific neuronal circuits to examine two behavioral paradigms, the Drosophila models of pain and fear, that the investigator's group has developed. Both of these involve basic neuronal elements, including sensors that detect the stimulus and neuronal networks that execute the behavior when the sensor is activated.
Using Drosophila as a model organism, this research will increase the scientific understanding of the mechanisms that allow complex behaviors to be encoded by the genome,. The basic principles learned from these studies will provide a foundation for understanding the molecular and neural principles that underlie behavior in humans and other organisms. It is expected that students at a range of levels, from high school to graduate school, will participate in this research.

This project is to investigate the role of genes in aging, using Drosophila as a model system, in which the genes can be manipulated in various ways. One way is to screen for mutant genes that extend lifespan, as exemplified by the mutant methuselah, isolated in our laboratory. Since increased longevity is usually correlated with enhanced resistance to various forms of stress, screening for stress-resistant mutants is another approach to identifying life-assurance genes. A third approach is to identify genes whose expression is strongly increased in response to stress, then to determine whether overexpression of some such genes can extend lifespan. All these methods have provided candidate genes related to life extension. Chosen ones will be analyzed by molecular techniques to identify their tissue specificity of expression and their signal transduction and other cellular processes underlying aging, thus providing clues to roles in manipulation of specific gene expression that can contribute to enhanced, healthy lifespan.

DESCRIPTION (provided by applicant): Caloric restriction (CR), a decrease in nutritional level without causing malnutrition, is well known to increase lifespan across species, from yeast to rodents. Ad libitum feeding can therefore be viewed as having a toxic effect on lifespan. Indeed, overnutrition in humans is one of the leading risk factors for age-related diseases and mortality. We propose to characterize the effects of overnutrition in Drosophila, and seek ways to mitigate them by finding mutants that show either enhanced sensitivity or resistance to overnutrition. The identification of molecular pathways involved should provide tools in the understanding of this phenomenon, by leading us to the downstream genes that manifest the lifespan changes. Examination of the pathological and physiological changes resulting from overnutrition in normal and mutant flies will cast light on their involvement in aging and age-related diseases. Most investigations have been directed at the effects of undernutrition as a beneficial factor in extending lifespan. This proposal represents an alternate approach, focusing on overnutrition to exaggerate deleterious effects, thus providing a sensitized system in which to discover methods of ameliorating them.

DESCRIPTION (provided by applicant): Caloric restriction (CR), a decrease in nutritional level without causing malnutrition, is well known to increase lifespan across species, from yeast to rodents. Ad libitum feeding can therefore be viewed as having a toxic effect on lifespan. Indeed, overnutrition in humans is one of the leading risk factors for age-related diseases and mortality. We propose to characterize the effects of overnutrition in Drosophila, and seek ways to mitigate them by finding mutants that show either enhanced sensitivity or resistance to overnutrition. The identification of molecular pathways involved should provide tools in the understanding of this phenomenon, by leading us to the downstream genes that manifest the lifespan changes. Examination of the pathological and physiological changes resulting from overnutrition in normal and mutant flies will cast light on their involvement in aging and age-related diseases. Most investigations have been directed at the effects of undernutrition as a beneficial factor in extending lifespan. This proposal represents an alternate approach, focusing on overnutrition to exaggerate deleterious effects, thus providing a sensitized system in which to discover methods of ameliorating them.