We propose to investigate the molecular genetics of the bacterio-opsin (bop) gene and develop a genetic system for the expression of site-directed mutations of the bop gene. These investigations will complement the experimental efforts of Dr. R. Stroud's laboratory at UCSF on the structural analysis of bacterio-opsin, bacteriorhodopsin and purple membrane. We will adapt the bop gene of H. halobium into an E. coli expression vector to examine the compatibility of the bacterio-opsin protein with E. coli. If expression is successful, it can be purified and reconstituted with lipids and retinal to form a functional membrane vesicle. We will also attempt to develop a transformation and vector system for H. halobium which eventually can be used for bop gene expression. Primarily by recombinant DNA and sequencing technologies we intend to compare the WT and mutant bop genes of H. halobium along with the bop genes of other purple membrane Halobacteria for protein and nucleotide sequence polymorphisms. Mutations to bop- occur at frequencies of 10 to mthe minus 4 and will be used to define the boundaries for the transcriptional elements of the bop gene. These mutations are almost exclusively associated with putative insertions of DNA in or near the bop gene. The apparent genomic hypervariability of H. halobium will be investigated by characterizing these putative insertions and determing their distributions in WT H. halobium and its mutant genomes. Investigations of the molecular genetics of the bop gene of H. halobium will provide the first insight into the genome organization and genetic mechanisms of the archaebacteria. The distinct phylogenetic position of the archaebacteria relative to procaryotic and eucaryotic forms of life dictate the possibility of novel genetic mechanisms which could provide new insignts to further our understanding of knwon genetic mechanisms. In particular, H. halobium is known to incur insertions, deletions, and rearrangement od DNA segments which are associated with phenotypic variations that occur in unusually high frequencies (10 to the minus 2 to 10 to the misus 4).
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