A cloned fragment of spinach chloroplast DNA carrying the gene for the large subunit of ribulose bisphosphate (RuBP) carboxylase has been analysed by electron microscopy of R-loops, by hybridization to Northern blots of chloroplast RNA, by S1 nuclease mapping and by DNA sequencing. The transcribed region of the gene is 1690 +/- 3 nucleotides long and co-linear with its mRNA. It comprises a 178-179 bp 5' untranslated sequence, a 1425 bp coding region and an 85-88 bp 3' untranslated region. The deduced sequence of the 475 amino acids of the spinach large subunit protein shows 10% divergence from that of the maize large subunit protein (1). The nucleotide sequence divergence between spinach and maize over the same coding region is 16% but in the transcribed flanking regions it is 35%. Features of the spinach chloroplast gene which resemble those of bacterial genes include a 5-base Shine-Dalgarno sequence complementary to a sequence near the 3' end of chloroplast and bacterial 16S rRNA, a promoter region partially homologous to a consensus sequence of bacterial promoters, and a transcription termination region capable of forming a typical stem and loop structure.

Mutagenesis in vitro has been used to obtain mutant forms of the bifunctional enzyme, chorismate mutase/ prephenate dehydrogenase. Plasmid D N A containing the genes that code for the enzyme was treated with hydroxylamine and the resulting products were used to transform strains of Eschericliia coli. Two types of mutant were isolated. One contained enzyme which was niutase-positive, dehydrogenase-negative while the other did not exhibit either activity. Kinetic and physical analysis of one of the purified monofunctional enzymes showed that the loss of dehydrogenase activity was due to modification of the binding site for NAD. The results open the way for molecular studies of structure-function relationships with this bifunctional enzyme. A separate bifunctional enzyme chorismate mutase/prephenate dehydrogenase (EC 1.3.1.12), which is coded for by the tyrA gene, catalyses both the conversion of chorismate to prephenate, in the presence of NAD, and of prephenate to 4-hydroxyphenylpyruvate. These reactions form part of the tyrosine biosynthetic pathway. Chorismate mutase/prephenate dehydrogenase from both E. coli and Aerobacter aerogenes has been extensively studied. However, the kinetic approaches to the determination of the number of catalytic sites that are involved in the conversions of chorismate to prephenate and of prephenate to hydroxyphenylpyruvate have not yielded definitive answers [6,7]. Because of the successful isolation of monofunctional mutant forms of chorismate mutasef prephenate dehydrogenase from S. typhimurium [2,8] it seemed that more clear-cut results might be obtained by using the genetic approach for studies on the E. coli enzyme. T o facilitate the subsequent purification of mutant forms of chorismate mutasefprephenate dehydrogenase, mutagenesis was performed in vitro on a multicopy recombinant plasmid which carries the tyrA gene and which is responsible for markedly elevated yields of chorismate mutasefprephenate dehydrogenase [9]. This paper reports the isolation and subsequent characterization of chorismate mutase/prephenate dehydrogenase obtained from six mutants and evidence is presented to show Enzymes. Chorismate mutase, prephenate dehydrogenase (EC ~-1.3.1.12); chorismate mutase, prephenate dehydratase (EC 4.2.1.51). In the phenylalanine biosynthetic pathway in