Isolation of a cDNA Clone for the γ Subunit of the Chlamydomonas Reinhardtii CF1
A cDNA library from Chlamydomonas reinhardtii was probed with antiserum directed against the nuclear encoded γ subunit of the chloroplast H+-translocating ATP synthase (CF0-CF1) of C. reinhardtii. A cDNA was isolated and transcribed in vitro. The transcript was translated in vitro and immunoprecipitated with anti- γ serum to yield a product that co-electrophoresed with the immunoprecipitated product from in vitro translated polyadenylated RNA. These proteins were larger than the mature γ subunit. Thus the γ subunit is synthesized as a precursor of greater molecular weight in C. reinhardtii, as has been suggested for the y subunit from spinach (Nelson et al., 1980).
The precursor protein encoded by this cDNA was imported into pea chloroplasts and processed to a lower molecular weight polypeptide that co-electrophoresed with mature C. reinhardtii γ subunit. The largest cDNA isolated is about the same length as the corresponding mRNA (about 2000 bases). Southern analyses revealed restriction fragment length polymorphisms and that the γ subunit is probably encoded by an intron-containing single copy gene.
KeywordsRabbit Reticulocyte Lysate Translation Reaction Detergent Solubilization Bethesda Research Laboratory Lower Molecular Weight Polypeptide
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