COX6b in rice has a long N-terminal sequence comparedwith those in other species

54. COX6b in rice has a long N-terminal sequence compared with those in other species K. OHTSU, S. HAMANAKA, M. NAKAZONO and A. HIRAI Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Science, The University ofTokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-3657 Japan Mitochondria of higher plants contain two respiratory pathways: the cyanide-sensitive cytochrome pathway, and the cyanide-resistant alternative pathway. Cytochrome c oxidase (COX) is a terminal oxidase in the cytochrome pathway that converts oxygen to H₂0. The COX of higher plants is composed of at least 10 subunits. The three largest subunits (COX1, COX2 and COX3) are encoded by the mitochondrial genome, and the remaining subunits are encoded by the nuclear genome. The mitochondnal-encoded COX genes have been extensively characterized in higher plants including rice (Schuster and Brennicke 1994). However, only a limited number of studies have been carried out on the nuclear-encoded COX genes of plants. It is required to accumulate more information about them. The gene for the human COX6b subunit has been described by Taanman et al. (1990) and Carrero-Valenzuela et al. (1991). The COX12 gene, which encodes the COX6b subunit of Saccharomyces cerevisiae has been described by LaMarche et al. (1992). The protein has a molecular size of approximately 10 kDa, and analysis of the COX12-disrupted strain indicated that the COX6b subunit is essential during assembly for full cytochrome c oxidase activity. So far, information about the COX6b subunit in plants has not been available. In this report, we characterized a novel gene encoding COX6b subunit in rice. As a first step in detecting novel COX genes in higher plants, we searched the rice EST clone database for genes that are homologous to human nuclear-encoded COX genes. As a result, the amino acid sequence of human COX6b protein was found to show a significant homology to the putative protein encoded by the EST clone S12878 from rice (Oryza sativa cv. Nipponbare) green seedlings. The 600 bp insert of the cDNA clone S12878 was completely sequenced. However, no putative initiation codon was included in the S12878 clone. Therefore the 5'RACE experiment was carried out using the Cap site cDNA derived from green leaves. We obtained PCR fragments and determined their complete nucleotide sequences. With these sequences, an open reading frame (ORF) could be identified. The ORF, which was designated as COX6b, encodes 169 amino acids with a calculated molecular mass of 19 kDa. A comparison of the deduced amino acid sequences of the COX6b precursor proteins from rice, human, cow and Saccharomyces showed that, surprisingly, the N-terminal portion of the rice COX6b precursor protein was extended (Fig. 1). We also sequenced a cDNA clone of an Arabidopsis gene that is homologous to human COX6b and yeast COX12 (data not shown). The deduced amino acid sequence of the Arabidopsis COX6b gene was also extended toward the N-terminus. Thus, it is likely that this feature may be common among higher plants. Although the structure of rice COX6b protein is unusual, the result of Northern blot analysis showed that in rice, this gene was constitutively expressed in various organs in several growth stages (Ohtsu et al. 1999). Therefore the rice COX6b gene we characterized here may produce functional COX6b protein in rice. References Carrero-Valenzuela, R.D., F. Quan, R. Lightowlers, N.G. Kennaway, M. Litt and M. Forte, 1991. Human cytochrome c oxidase subunit VIb: characterization and mapping of a multigene family. Gene 102: 229-236. LaMarche,A.E.P., M.I. Abate, S.H.P. Chan and B.L. Trumpower, 1992. Isolation and characterization of COX12, the nuclear gene for a previously unrecognized subunit of Saccharomyces cerevisiae cytochrome c oxidase. J. Biol. Chem. 267: 22473-22480 .Lightowlers, R.N. and R.A. Capaldi, 1989. Nucleotide sequence of the cDNA encoding subunit AED (VIB) of beef heart cytochrome c oxidase. Nucleic Acids Res. 17: 5845. Ohtsu, K., S. Hamanaka, K Yamazaki, M. Nakazono and A. Hirai, 1999. Characterization of a cDNA encoding a novel subunit for cytochrome c oxidase (COX6b) from rice. Breed. Sci. 49: 21 1-215. Schuster, W. and A. Brennicke, 1994. The plant mitochondrial genome: physical structure, information content, RNA editing, and gene migration to the nucleus. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45: 61-78. Taanman, J.-W., C. Schrage, N.J. Ponne, A.T. Das, P.A. Bolhuis, H. de Vries and E. Agsteribbe, 1990. Isolation of cDNAs encoding subunit VIb of human cytochrome c oxidase and steady-state levels of CoxVIb mRNA in different tissues. Gene 93: 285-291. Thompson, J.D., D.G. Higgins and T.J. Gibson, 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4763-4680.

54. COX6b in rice has a long N-terminal sequence compared with those in other species

K. OHTSU, S. HAMANAKA, M. NAKAZONO and A. HIRAI
Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Science, The University ofTokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-3657 Japan

Mitochondria of higher plants contain two respiratory pathways: the cyanide-sensitive cytochrome pathway, and the cyanide-resistant alternative pathway. Cytochrome c oxidase (COX) is a terminal oxidase in the cytochrome pathway that converts oxygen to H₂0. The COX of higher plants is composed of at least 10 subunits. The three largest subunits (COX1, COX2 and COX3) are encoded by the mitochondrial genome, and the remaining subunits are encoded by the nuclear genome. The mitochondnal-encoded COX genes have been extensively characterized in higher plants including rice (Schuster and Brennicke 1994). However, only a limited number of studies have been carried out on the nuclear-encoded COX genes of plants. It is required to accumulate more information about them.

The gene for the human COX6b subunit has been described by Taanman et al. (1990) and Carrero-Valenzuela et al. (1991). The COX12 gene, which encodes the COX6b subunit of Saccharomyces cerevisiae has been described by LaMarche et al. (1992). The protein has a molecular size of approximately 10 kDa, and analysis of the COX12-disrupted strain indicated that the COX6b subunit is essential during assembly for full cytochrome c oxidase activity. So far, information about the COX6b subunit in plants has not been available. In this report, we characterized a novel gene encoding COX6b subunit in rice.

As a first step in detecting novel COX genes in higher plants, we searched the rice EST clone database for genes that are homologous to human nuclear-encoded COX genes. As a result, the amino acid sequence of human COX6b protein was found to show a significant homology to the putative protein encoded by the EST clone S12878 from rice (Oryza sativa cv. Nipponbare) green seedlings. The 600 bp insert of the cDNA clone S12878 was completely sequenced. However, no putative initiation codon was included in the S12878 clone. Therefore the 5'RACE experiment was carried out using the Cap site cDNA derived from green leaves. We obtained PCR fragments and determined their complete nucleotide sequences. With these sequences, an open reading frame (ORF) could be identified. The ORF, which was designated as COX6b, encodes 169 amino acids with a calculated molecular mass of 19 kDa. A comparison of the deduced amino acid sequences of the COX6b precursor proteins from rice, human, cow and Saccharomyces showed that, surprisingly, the N-terminal portion of the rice COX6b precursor protein was extended (Fig. 1). We also sequenced a cDNA clone of an Arabidopsis gene that is homologous to human COX6b and yeast COX12 (data not shown). The deduced amino acid sequence of the Arabidopsis COX6b gene was also extended toward the N-terminus. Thus, it is likely that this feature may be common among higher plants. Although the structure of rice COX6b protein is unusual, the result of Northern blot analysis showed that in rice, this gene was constitutively expressed in various organs in several growth stages (Ohtsu et al. 1999). Therefore the rice COX6b gene we characterized here may produce functional COX6b protein in rice.

References

Carrero-Valenzuela, R.D., F. Quan, R. Lightowlers, N.G. Kennaway, M. Litt and M. Forte, 1991. Human cytochrome c oxidase subunit VIb: characterization and mapping of a multigene family. Gene 102: 229-236.

LaMarche,A.E.P., M.I. Abate, S.H.P. Chan and B.L. Trumpower, 1992. Isolation and characterization of COX12, the nuclear gene for a previously unrecognized subunit of Saccharomyces cerevisiae cytochrome c oxidase. J. Biol. Chem. 267: 22473-22480

.Lightowlers, R.N. and R.A. Capaldi, 1989. Nucleotide sequence of the cDNA encoding subunit AED (VIB) of beef heart cytochrome c oxidase. Nucleic Acids Res. 17: 5845.

Ohtsu, K., S. Hamanaka, K Yamazaki, M. Nakazono and A. Hirai, 1999. Characterization of a cDNA encoding a novel subunit for cytochrome c oxidase (COX6b) from rice. Breed. Sci. 49: 21 1-215.

Schuster, W. and A. Brennicke, 1994. The plant mitochondrial genome: physical structure, information content, RNA editing, and gene migration to the nucleus. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45: 61-78.

Taanman, J.-W., C. Schrage, N.J. Ponne, A.T. Das, P.A. Bolhuis, H. de Vries and E. Agsteribbe, 1990. Isolation of cDNAs encoding subunit VIb of human cytochrome c oxidase and steady-state levels of CoxVIb mRNA in different tissues. Gene 93: 285-291.

Thompson, J.D., D.G. Higgins and T.J. Gibson, 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4763-4680.