Using the facultative phototrophic bacterium, Rhodopseudomonas sphaeroides, we are attempting to understand, at the molecular level, gene control of the inducible photosynthetic membrane system (ICM). The genetic basis for ICM expression extends to the: 1. Major bacteriochlorophyll-protein spectral complexes, cytochromes, ATPase and other ICM-associated enzyme activities and structural proteins, 2. The pathways for carotenoid and bacteriochlorophyll synthesis, 3. Phospholipid biosynthetic enzymes and the movement of phospholipids, and 4. The regulatory systems resulting in cellular adaptation to changes in oxygen partial pressure and light intensity, and those mechanisms involved in assembly in an orderly manner, as well as the structure-function interrelationships of the ICM. It is proposed that we identify, clone and characterize those genetic regions relevant to ICM structure and regulation. This study includes, structure of the DNA region, through restriction endonuclease analysis, DNA sequencing and in vitro and in vivo characterization of gene expression. We propose to develop procedures for the selection, isolation and characterization, both in vivo and in vitro, of mutations which are relevant to ICM structure and function by employing synthetic deoxyoligonucleotide and transposon induced mutagenesis. We intend to derive genetic systems to monitor the expression of these cloned regions in both homologous and heterologous hosts in order to better understand the mechanisms of gene control. Such studies will bear directly on the broader question of the nature of the regulatory signals and their function in heterologous host systems. Finally, we intend to develop genetic and pseudogenetic systems for the movement, complementation, expression and facile handling of genetic regions in R. sphaeroides. We shall attempt to develop conjugation and transduction systems, cosmids, plasmids, promoter cloning vehicles, etc., employing classical procaryotic genetic techniques as well as recombinant DNA technologies. Understanding the functional and structural implications of photosynthesis is fundamental to the adequate nutrition and, therefore, health of human populations. Of equal importance, the structure-function interrelationships of membrane-associated activities is of critical health importance because of the nature of health related membrane phenomena, e.g. membrane receptors, mitochondrial function, excretion, lipoprotein structure and function, etc.
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