86] Isolation and characterization of genes for cytochrome [formula omitted] complex

Abstract

This chapter describes procedures used for the isolation and preliminary characterization of genes encoding the cytochrome b 6 /f complex from the filamentous cyanobacterium Nostoc PCC 7906. The proton-translocating thylakoid membrane cytochrome b 6 /f complex catalyzes electron transfer between the two photosystems (PS) and is required for cyclic electron flow around photosystem I (PSI). In cyanobacteria as in plant chloroplasts, the complex consists of four essential polypeptides: cytochromes f and b 6 , the Rieske Fe-S protein, and a subunit IV. In the cyanobacterium Anabaena these have apparent molecular weights of 31,000, 22,500, 22,000 and 16,000, respectively. Cloned genes can now be introduced by transformation or conjugation into several strains of cyanobacteria enabling, among other applications, a molecular genetic analysis of the structure and function of thylakoid membrane protein complexes. An additional requirement for this approach is the availability of cloned genes for these proteins and the determination of their nucleotide and derived amino acid sequences. Heterologous hybridization (the use of a gene from one source as a probe for isolating a gene from another source) was the approach first used to isolate cyanobacterial genes and perhaps remains the most widely used one. Its success ultimately depends on the level of nucleotide sequence similarity between the probe and the target DNA.