Phycocyanin (PC) is a light-harvesting protein common to blue-green and red algae. We have isolated the genes for the two apoprotein subunits, a and f, of PC from the blue-green alga Agmenellum quadruplicatum In most photosynthetic organisms the major light-harvesting pigments are cyclic tetrapyrroles bound noncovalently to polypeptides intrinsic to the photosynthetic membrane. Blue-green algae (cyanobacteria) and red algae follow this pattern but, in addition, have large amounts of antenna pigments in the form of linear tetrapyrroles covalently bound to water-soluble polypeptides. These proteins, termed phycobiliproteins, occur as three major types: phycoerythrin, phycocyanin (PC), and allophycocyanin (1). Each phycobiliprotein consists of two subunits, a and /3, which contain characteristic numbers and types of chromophores. Phycobiliproteins aggregate to form complexes called phycobilisomes (1). The assembly of phycobilisomes is mediated by nonpigmented linker polypeptides. These linkers also alter the spectral properties of phycobiliproteins so as to ensure an efficient transfer of absorbed light energy to the membrane (2). Phycobilisomes, in turn, are found attached to the outer surface of the thylakoid membrane, perhaps in association with photosystem 11 (3).Our aim is to describe the structure of the phycobilisome and the regulation of the genes encoding its components.These studies may also shed light on the evolution of Ohycobiliprotein genes. As a first step, we have cloned and sequenced the genes encoding the a and ,8 subunit apoproteins of PC from the blue-green alga Agmenellum quadruplicatum. A preliminary report of these results has been presented (4).
MATERIALS AND METHODSDNA purification. A. quadruplicatum strain PR-6 was cultivated axenically in medium A with NaNO3 (1 mg/ml) as described (5). Cells were harvested before reaching a density of 5 x 107 cells per ml, washed in 10% sucrose/50 mM Tris HCl, pH 8.0/100 mM Na2EDTA, and stored at -80°C. Lysis was achieved by thawing, adding egg-white lysozyme to a final concentration of 10 mg/ml, incubating at 37°C for 30 min, and adding N-lauroyl sarcosine (10% wt/vol stock solution) to a final concentration of 1%. An equal amount (wt/vol) of CsCl was added and DNA was purified by buoyant-density centrifugation. DNA-containing fractions were recentrifuged in the presence of ethidium bromide at 150 ,ug/ml. The dye was removed by n-butanol extraction and the DNA was dialyzed against 10 mM Tris-HCl, pH 8.0/1 mM Na2EDTA.RNA Purification. An exponentially growing culture of A. quadruplicatum was harvested, resuspended in the original volume of fresh medium A lacking nitrate, and incubated with aeration and illumination as before. The A620/A680 ratio decreased from 0.9 to 0.3 within 24 hr, whereupon the cells were harvested, resuspended in medium A with nitrate (1 mg/ml) and incubated as before. After 8 hr, cells were harvested by centrifugation and resuspended in 10 mM Tris HCl, pH 8.0/1 mM Na2EDTA. Cells were lysed by passage through a French pressu...