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).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031785-03
Application #
3280100
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1983-06-01
Project End
1986-05-31
Budget Start
1985-06-01
Budget End
1986-05-31
Support Year
3
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Turner, G J; Reusch, R; Winter-Vann, A M et al. (1999) Heterologous gene expression in a membrane-protein-specific system. Protein Expr Purif 17:312-23
Turner, G J; Miercke, L J; Mitra, A K et al. (1999) Expression, purification, and structural characterization of the bacteriorhodopsin-aspartyl transcarbamylase fusion protein. Protein Expr Purif 17:324-38
Gropp, F; Gropp, R; Betlach, M C (1995) Effects of upstream deletions on light- and oxygen-regulated bacterio-opsin gene expression in Halobacterium halobium. Mol Microbiol 16:357-64
Shand, R F; Betlach, M C (1994) bop gene cluster expression in bacteriorhodopsin-overproducing mutants of Halobacterium halobium. J Bacteriol 176:1655-60
Turner, G J; Miercke, L J; Thorgeirsson, T E et al. (1993) Bacteriorhodopsin D85N: three spectroscopic species in equilibrium. Biochemistry 32:1332-7
Mitra, A K; Miercke, L J; Turner, G J et al. (1993) Two-dimensional crystallization of Escherichia coli-expressed bacteriorhodopsin and its D96N variant: high resolution structural studies in projection. Biophys J 65:1295-306
Gropp, R; Gropp, F; Betlach, M C (1992) Association of the halobacterial 7S RNA to the polysome correlates with expression of the membrane protein bacterioopsin. Proc Natl Acad Sci U S A 89:1204-8
Miercke, L J; Betlach, M C; Mitra, A K et al. (1991) Wild-type and mutant bacteriorhodopsins D85N, D96N, and R82Q: purification to homogeneity, pH dependence of pumping, and electron diffraction. Biochemistry 30:3088-98
Shand, R F; Betlach, M C (1991) Expression of the bop gene cluster of Halobacterium halobium is induced by low oxygen tension and by light. J Bacteriol 173:4692-9
Thorgeirsson, T E; Milder, S J; Miercke, L J et al. (1991) Effects of Asp-96----Asn, Asp-85----Asn, and Arg-82----Gln single-site substitutions on the photocycle of bacteriorhodopsin. Biochemistry 30:9133-42

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