CHARACTERIZATION AND GENOMIC PARTITIONING OF CHLOROPLAST RIBOSOMAL PROTEINS FROM HIGHER PLANTS (NICOTIANA, TABACUM).

Abstract

Chloroplast and cytoplasmic ribosomes have been isolated from a number of species of the angiosperm genus Nicotiana. Ribosomal subunit and monosome proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Resultant two-dimensional electrophoretic patterns of chloroplast and cytoplasmic ribosomal proteins were processed by a computer algorithm, developed to formally compare different electrophoretic patterns by the construction of two-dimensional, conformal average electrophoretic mobility maps. The chloroplast ribosomal subunit of N. tabacum contains 22-24 distinct basic polypeptides (pI > 5) and 2-3 acidic proteins (pI < 5). The 50S chloroplast ribosomal subunit possesses at least 1 acidic and 33-35 basic proteins. 40S and 60S cytoplasmic ribosomal subunits of the same species have 26-30 and 47-50 basic polypeptides, respectively. Molecular weights of chloroplast ribosomal proteins (ChRP) and cytoplasmic ribosomal proteins (CyRP) were estimated. There was little similarity between the 2D electrophoretic patterns of ChRP and CyRP of N. tabacum. However, 2D-PAGE patterns of N. tabacum ChRP and CyRP were qualitatively isomorphous with homologous patterns of Chlamydomonas reinhardi, pea and spinach. In terms of molecular weight and electrophoretic pattern N. tabacum ChRP were found to be more closely affiliated with prokaryotic ribosomal proteins than with CyRP from the same species. ChRP were isolated from N. gossei (an Australian species) and its reciprocol interspecies hybrids with N. tabacum (denoted by: T x G and G x T). Interspecies polymorphisms between homologous N. tabacum and N. gossei ChRP were delineated by computerized mobility mapping and co-electrophoresis of radiolabeled N. tabacum ChRP with a large molar excess of N. gossei ChRP. The inheritance mode (Mendelian vs. maternal) of a number of well-defined interspecies ChRP polymorphisms was determined by co-electrophoresis of radioiodinated N. tabacum ChRP with T x G and G x T hybrid ChRP. Results indicate that at least 4 30S ChRP and 3 50S ChRP are encoded by nuclear genes. 30S ChRP from an N. tabacum line carrying a maternally-inherited streptomycin-resistance mutation (SR-1) were compared to N. tabacum 30S ChRP by mobility mapping. Two differences were established between the SR-1 and wild-type 30S ChRP average mobility maps. These findings correlate with the reduced affinity of SR-1 30S chloroplast ribosomal subunits for ('3)H-dihydrostreptomycin, and show that at least one 30S ChRP is encoded by chloroplast DNA. Preparative 2D-PAGE and reverse high performance liquid chromatography (RPHPLC) separation techniques for complex ribosomal protein mixtures were developed. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI