Localization of genes for coupling factor subunits on the spinach plastid chromosome

Abstract

1) Messenger RNA obtained from spinach cotyledons directs the synthesis of all five CF1 subunits in vitro in a rabbit reticulocyte translation system. The alpha, beta and epsilon subunit polypeptides were found as translation products from ptRNA and whole-cell poly A(-)-RNA. The gamma and delta subunits were synthesized from whole-cell poly A(+)-RNA as precursors of substantially greater molecular weight indicating that they originate in the nucleus and are imported into the chloroplast. High resolution electrophoresis, immunoprecipitation with antibodies against individual CF1 subunits (Nelson et al. 1980), and proteolytic peptide mapping were employed to identify the products. 2) The genes for alpha, beta and epsilon subunits of CF1 were located by hybrid-selected translation with matrix-immobilized ptDNA fragments of known map position. The genes for all three CF1 subunit polypeptides are located in the large single-copy segment (cf. Herrmann et al. 1980b) of the circular ptDNA and each gene appears to be present once on the chromosome. The genes for the beta and epsilon subunits lie near each other in immediate vicinity to the structural gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase. The gene for the alpha subunit is separated by approximately 40 kbp from this gene cluster, and located near the gene for the 32 kd photosystem II polypeptide (Driesel et al. 1980). 3) Restriction fragments of spinach ptDNA with CF1 subunit genes were cloned into pBR 322 and used to construct detailed maps.