PHYSIOLOGY OF BACTERIO-OPSIN GENE EXPRESSION IN HALOBACTERIUM

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Bacteriorhodopsin is an integral membrane photoreceptor found in Halobacterium halobium, an extremely halophilic member of the domain Archea. It possesses near structural identify to rhodopsin, a photoreceptor integral to the vision process in animals. The long term goal of this research is to elucidate the regulatory circuitry involved in bacterio-opsin (bop) gene expression. H. halobium occupies unique environmental and evolutionary niches, and elucidation of new regulatory circuitry in this organism may provide new models for genes in eukaryotes and eubacteria in which the regulatory mechanism(s) are unknown. One aim is to use the techniques, methodologies and experimental design of this project to train Native American Minority Biomedical Students in a variety of relevant and current biomedical skills. Students will have the opportunity to learn how to (i) grow and manipulate both H. halobium and E. coli; (ii) quantitate growth spectrophotometrically and by viable counts; (iii) isolate plasmids and prepare 32P riboprobes; (iv) isolate RNA and probe Northern blots to quantitate bop gene cluster mRNA levels; (v) isolate and quantitate bacteriorhodopsin protein spectrophotometrically, etc. In addition to learning various techniques, students will learn how to design their own experiments and analyze the data generated from these experiments. The results of the research carried out by the student will be presented by the student at regional and national meetings, and if possible, be published in a peer-reviewed journal. A second aim is to quantitate mRNA levels in a series of experiments that will: (i) determine the order in which bop, brp and bat transcripts appear when induced both by light and by low oxygen tension, (ii) test the hypothesis that continued exposure to high levels of dissolved oxygen will suppress induction of the bop gene cluster, and (iii) determine if the bop gene cluster is induced by other environmental conditions such as amino acid starvation. A third aim is to determine the hierarchical regulatory relationships among the bop, brp and bat genes. This will be done by placing mutants that contain mutations in one of the genes under inducing conditions and quantitating transcript levels from the other two genes. A fourth aim is to construct deletion mutations in the chromosomal genes of the bop gene cluster. Deletion mutations will provide unambiguous genetic backgrounds in which to evaluate physiological experiments and will be suitable for genetic complementation studies.