Nucleotide Sequence for the Genomic DNA Encoding the Chloroplast ATP Synthase γ-Subunit Gene (atpC) from Chlamydomonas reinhardtii

Abstract

The ATP synthase complex (CFo-CF1) in chloroplast thylakoid membranes couples the chemical potential energy in the protonmotive force to the synthesis of ATP. A unique feature of the chloroplast enzyme is that the catalytic activity is partially regulated by the oxidation state of the CF, ysubunit (2, 4). The a-subunit contains two cysteine residues that are speculated to form an intrapeptide disulfide bridge (5). The oxidation of the vicinal sulfhydryls together with the reduction of the ensuing disulfide is believed to act as a redox regulatory switch (2. 4). The eukaryotic green alga Chlamvl,,dornonas reinhardtli can grow exclusively on acetate, which allows one to isolate conditionally lethal mutant strains defective in photosynthesis. This makes C. reinhardtii an ideal model system for the application of both classical and modern molecular genetics. Although the cDNA for the C. reinhardtii CF, --subunit gene has been cloned and sequenced (6), in order to identify mutations in the nuclear gene (atpC), it was necessary to obtain the genomic DNA sequence (7). Furthermore, the genomic atpC gene can be used to rescue atpC mutants and to generate and tag other types of mutations in C reinhardtil. The atpC gene was obtained from genomic DNA by using DNA primers corresponding to the 5' and 3' ends of the cDNA and polymerase chain reaction technology to amplify the intervening sequence (Table I). The nucleotide and deduced amino acid sequences are shown in Figure 1. The atpC gene contains four introns. The first intron, only 85 bases long, resides very close to the transit peptide/mature peptide cleavage site. It is interesting to note that the third intron in the C. reinhardtil gene corresponds exactly to the position of the second intron in the spinach -y-subunit genomic DNA (3). All four introns are located in the 5' portion of the gene while the sequence encoding the putative regulatory cysteines are located in the 3' portion. This is in contrast to the spinach sequence in which the DNA encoding the two cysteines are located between the two introns (3). The exon-intron boundaries for the first two introns and the 5' junction of the third intron coincide with the AG:GT motif which is the consensus splice junction in plants (1). However, the 3' junction for the third intron and both junctions for the fourth intron do not have the consensus sequence. The four introns do not have any significant sequence identity with each other and have approximately the same G-C content as the coding region.