Nucleotide Sequence of ATPase Subunit 6 Gene of Maize Mitochondria

Abstract

The ATPase subunit 6, located in the inner mitochondrial membrane, is encoded by mitochondrial genomes in animals and fungi. We have isolated and charcterized a mitochondrial gene, designated atp 6, that encodes the subunit 6 polypeptide of Zea mays. Nucleotide and predicted amino acid sequence comparisons have revealed a homology of 44.6 and 33.2% with the yeast ATPase subunit 6 gene and polypeptide, respectively. The predicted protein in maize contains 291 amino acids with a molecular weight of 31,721. Hydropathy profiles generated for the maize and yeast polypeptides are very similar and contain large hydrophobic domains, characteristic of membrane bound proteins. RNA transfer blot analysis indicates that atp 6 is actively transcribed. Interestingly, 122 base pairs of nucleotide sequence interior to atp 6 have extensive homol-ogy with the 5' end of the cytochrome oxidase subunit II gene of maize mitochondru, suggesting recombination between the two genes. The mt2 ATPase complex, located in the inner mt membrane, consists of three components designated Fo, F,, and the oligo-mycin-sensitivity-conferring protein (OSCP) (27). The various subunits making up the complex are encoded either by the nuclear or mt genomes. In yeast, subunits 6, 8, and 9 of the Fo component are mt gene products while the other subunits are of nuclear origin (16, 27, 28). Animal systems and certain fungi differ in that subunit 9 is encoded within the nucleus (25). Higher plant mt genomes contain a gene coding for ATPase subunit 9 (8), yet differ from both animals and fungi in that they also code for the alpha subunit of the F, component (4, 1 1). Two different methods have been used to identify protein encoding genes ofthe maize mt genome. The Cyt oxidase subunit II and apocytochrome b genes were located with heterologous probes of the corresponding genes from Saccharomyces cerevi-siae and Kluyveromyces lactis, respectively (7, 9). The other approach involved the isolation and sequencing of an actively transcribed clone selected from a mtDNA library, followed by computer searches of gene banks to identify the gene encoded by the clone. The ATPase subunit 9 gene of maize mitochondria was identified in this manner (8). Using the latter method, we have isolated and identified the maize mt F0-ATPase subunit 6(s); nt, nucleotides; bp, base pairs gene. We present the nucleotide sequence of the subunit 6 gene and evidence that it is actively transcribed. MATERIALS AND METHODS Isolation of Nucleic Acids. Mitochondrial DNA and RNA were isolated from 6 to 7 d old dark-grown seedlings of Zea mays L, Wl82BN cms-SC or B73 cms-Tas previously described (21, 24). The cms-SC cytoplasm is a member of the T (Texas) group of male-sterile cytoplasms (10). Construction of Mitochondrial DNA Library. BamHI digests of total maize mtDNA were ligated into the plasmid vector pUC 8 (29), and transformed into Escherichia coli strain JM 83. Ampicillin-resistant, lac-colonies were selected, replicated and fixed onto nitrocellulose filters (17). Radioactive Labeling of DNA and RNA. Double-stranded DNA was labeled with [a-32P]dATP (NEN, 3200 Ci/mmol) by nick translation (22). Single-stranded DNA clones in bacterio-phage M 13 were labeled using the back priming technique ofHu and Messing (13). Total mtRNA was 5' end-labeled with [-32P] ATP (ICN, 7000 Ci/mmol) using T4 polynucleotide kinase (18). Gel Electrophoresis and Nucleic Acid Hybridizations. DNA fragments were separated by electrophoresis on 0.8% agarose gels in TPE buffer (80 mm Tris-phosphate, 8 mM EDTA (pH 7.8]) and transferred to nitrocellulose according to Wahl et al. (30). MtRNA was heat denatured and fiactionated by electrophoresis in 1.2% agarose gels containing 6% formaldehyde and blotted to nitrocellulose as described by Thomas (26). The 18S (1986 nt) and 26S (3546 nt) ribosomal RNAs of maize mitochondria were used as markers for estimating RNA sizes. All nucleic acid hybridizations were performed under conditions previously described (8). DNA Sequence Analysis. Cloning for sequence analysis was carried out using M13 bacteriophage vectors mplO and mp Il (18). Ligation and transformation procedures were as outlined by New England Biolabs. DNA sequences were determined by the chain-termination method of Sanger et al. (23) with a universal primer (PL Biochemicals). Sequencing gels were either 6 or 8% polyacrylamide and 0.4 mm thick. The sequencing strategy is shown in Figure 1. Sequence analyses were performed with computer programs furnished by Bionet or with a dot matrix computer program provided by M. Edgell (University of North Carolina, Chapel Hill). RESULTS Identification and Analysis of the Maiz ATPase Subunit 6 Gene. To locate mtDNA clones actively involved in transcription , end-labeled mtRNA was hybridized to a BamHi mtDNA 914