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The halo-opsin gene. I. Identification and isolation
P Hegemann1, A Blanck, H Vogelsang-Wenke
1Max-Planck Institut für Biochemie, D-8033, Martinsried, FRG.
The EMBO Journal
|January 1, 1987
Summary
Researchers purified halorhodopsin (HR), a light-driven chloride pump, and sequenced peptides to identify its halo-opsin (HO) structure. They then screened a gene bank, successfully isolating a clone encoding part of the HO gene.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- Halorhodopsin (HR) functions as a light-driven chloride pump in halobacteria.
- Understanding HR's structure is crucial for elucidating its mechanism.
Purpose of the Study:
- To determine the structure of halo-opsin (HO), the protein component of HR.
- To identify the gene encoding HO in Halobacterium halobium.
Main Methods:
- Purification of HR in native and denatured states.
- Chemical cleavage of HR into peptide fragments.
- Peptide sequencing using liquid-phase methods.
- Screening of a Halobacterium halobium cosmid gene bank using synthesized DNA probes.
- Subcloning and nucleotide sequencing of positive clones.
Main Results:
- Approximately 20% of the HO structure was determined from isolated peptide fragments.
- A cosmid clone (pAB H47) containing part of the HO gene was identified.
- Nucleotide sequencing confirmed the presence of the HO gene fragment within the clone.
Conclusions:
- The study successfully elucidated a portion of the halo-opsin structure.
- The gene encoding a significant part of halo-opsin was isolated from Halobacterium halobium.
- This provides a foundation for further genetic and structural studies of halorhodopsin.