Mapping of tRNA Genes on the Circular DNA Molecule of Spinacia oleracea Chloroplasts

Abstract

Chloroplasts contain their own complement of tRNAs, which are different from those of the cytoplasm and those of the mitochondria (Weil et al. 1977). The chloroplast tRNA structure is similar to that of prokaryotic tRNAs, at least for the two chloroplast tRNAs whose nucleotide sequences have been determined (Chang et al. 1976; Guillemaut and Keith 1977), and they are coded for by chloroplast DNA (Tewari and Wildman 1970; Gruol and Haselkorn 1976; Haff and Bogorad 1976; McCrea and Hershberger 1976; Schwartzbach et al. 1976).

The genes coding for chloroplast rRNAs have been mapped on higher plant and algal chloroplast DNAs. In maize and spinach, the chloroplast rRNA genes are located on two inverted repeats (Bedbrook et al. 1977; Whitfeld et al. 1978); in Euglena, the chloroplast rRNA genes exist as three clustered tandem repeats (Gray and Hallick 1978; Rawson et al. 1978). But until now, the chloroplast tRNA genes have not been localized. In this paper we describe the fractionation and identification of spinach (Spinacia oleracea) chloroplast tRNAs and the first results of our efforts to map the tRNA genes on spinach chloroplast DNA (a circular molecule of ~ 90 × 10⁶ daltons).

Unbroken Chloroplasts (Herrmann et al. 1975) were used in the preparation of tRNAs, aminoacyl-tRNA synthetases, and high-molecular-weight DNA. The tRNAs were prepared by phenol treatment of detergent-lysed chloroplasts, precipitated by ethanol, dissolved in 1 M NaCl, diluted to 0.2 M NaCl, incubated with DNase, and purified on DEAE-cellulose columns (Burkard et al. 1970). Aminoacyl-tRNA synthetases were obtained...