Genomic organization and sequence analysis of the cytochrome oxidase subunit II gene from normal and male-sterile mitochondria in sugar beet

Abstract

We have cloned and sequenced the cytochrome oxidase subunit II (coxII) gene from both normal and cytoplasmic male-sterile (CMS) sugar beet. The normal coxII (designated NcoxII) locus was found to be located 1491 bp upstream from the gene for cytochrome oxidase subunit I (coxI) on the same DNA strand and to have a 1463 bp intron which split the coding sequence into two exons (382 and 398 bp). The COXII protein contains 260 amino acid residues. We have also found two copies of the coxII gene (ScoxII-1 and ScoxII-2) to be present in the CMS genome. Our results suggest that the NcoxII gene diverges completely from the ScoxII-1 and ScoxII-2 genes 50 bp 5' to the ATG start codon. In addition, the ScoxII-1 and ScoxII-2 sequences could be readily discriminated from each other by the 3' end and the immediately adjacent flanking sequences of the gene: the 3' divergence results in a 101 codon extension of the ScoxII-2 ORF. Northern blot analysis demonstrates that the coxII gene exhibits altered transcript patterns in CMS compared with normal sugar beet. Different genomic arrangements of the coxII gene are considered to be the result of extensive intra- and inter-molecular recombination events involving the repeated DNA elements in the mitochondrial genome.

The cytoplasmic male-sterile type and normal type mitochondrial genomes of sugar beet share the same complement of genes of known function but differ in the content of expressed ORFs

“…Moreover, no additional proteins were detected in the Owen cytoplasm and no others were missing (Hallden et al 1992; M. P. Yamamoto, T. Kubo and T. Mikami, unpublished data). Taken together, these findings make Senda et al (1991) b Kubo and Mikami (1996) c Onodera et al (1999) it reasonable to consider that Owen CMS is not a lossof-function mutant but a gain-of-function mutant, and that the ORF encoding the 35-kDa protein is the best candidate for the underlying cause of Owen CMS. The sizes of the predicted translation products of Satp6 presequence, Scox2-2 and Sorf324 are 43.5, 39.9 and 36.3 kDa, respectively, which are close to the size of the novel 35-kDa protein.…”

Phylogenetic relationships of the mitochondrial genomes in the genus Glycine subgenus Soja.

“…The membranes were washed twice with 6 M urea, 0.4% SDS and 0.5 × SSC for 20 min at 42°C, and twice with 2 × SSC for 5 min at room temperature. The membranes were used to expose X-ray films for 1-4 h. Five clones of mtDNA fragments were used as probes; a 1.5-kb EcoRI fragment that contained cox1 (a gene encoding subunit 1 of cytochrome oxidase) from sugar beet (Senda et al, 1991), a 0.7-kb NcoI-HindIII fragment that contained cox2 (a gene encoding subunit 2 of cytochrome oxidase) from soybean (Kato, 1996), a 0.7-kb StyI fragment that contained atp6 (a gene encoding subunit 6 of F 0 -ATPase) from Oenothera (Schuster and Brennicke, 1987), a 0.7-kb XhoI-EcoRV fragment that contained atp9 (a gene encoding subunit 9 of F 0 -ATPase) from pea (Morikami and Nakamura, 1987), and a 0.7-kb EcoRIBamHI fragment that contained atp1 (a gene encoding subunit 1 of F 1 -ATPase) from sugar beet (Senda et al, 1993). Phylogenetic analysis.…”

The 5′-leader sequence of sugar beet mitochondrial atp6 encodes a novel polypeptide that is characteristic of Owen cytoplasmic male sterility

“…The GST-preS-FL fusion protein was further purified electrophoretically from gel slices using an Electro eluter Model 422 (BioRad). Antisera directed against SORF119 (aORF119; Satoh et al 2004), SCOXII-2 (aSCOXII-2; Senda et al 1991), and the N-terminal and C-terminal sequences of preSATP6 (apreS-N and apreS-C; Onodera et al 1999) were raised by using as immunogens synthetic oligopeptides coupled to the carrier protein keyhole limpet hemacyanin through MBS linking. The sequences of the synthetic peptides were as follows: NH 2 -LE-KLKQKTEALLKNVWNNYC-COOH (ORF119), NH 2 -(GC)ETATAEKRAKILEGMRHIAS-COOH (SCOX-II-2), NH 2 -MITKSKKEMIAYAK(C)-COOH (preS-N) and…”

“…The following three regions were inserted into the vector pGEX4T-1 (Amersham-Biosciences) and expressed as GST (glutathione S-transferase) fusion proteins: the C-terminal 44 amino acids of the predicted COXI protein (Senda et al 1991), the C-terminal 183 amino acids of the predicted SORF324 protein (Kubo and Mikami 1996) and the entire 387 amino acids of the predicted SATP6 leader peptide (preS-FL) (Onodera et al 1999). The GST-COXI and GST-ORF324 fusion proteins were purified using glutathione Sepharose 4B (AmershamBiosciences).…”