46. The chloroplast-encoded ORF39 sequence of Oryza rufipogon

2) Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, U.S.A.

We present here the nucleotide sequences of the chloroplast-encoded ORF39 (Herrmann et al. 1984) from Oryza rufipogon L. (a wild variety of rice, IRRI accession number 103823) and Oryza sativa IR54 (an Indica cultivar). This is the first reported DNA sequence of O. rufipogon. In both rice varieties, the ORF39 sequence is included within a single 364 dp EcoRI fragment. These fragments were isolated and cloned as follows (Zhao et al. 1989). Total rice DNA was digested with EcoRI and fractionated on an 0.8% agarose gel. A prominent band corresponding to DNA fragments of approximately 360 bp was eluted from the ethidium bromide stained gel. The DNA was ligated to EcoRI-cut pUC13 and used to transform E. coli JM1O1 cells. Colony hybridization analysis was performed with total rice DNA to select for the high copy number EcoRI fragment. The nucleotide sequence of the cloncd DNA was determined. The sequences were then entered into the Microgenie ^TM computer sequence software (Beckman Instruments, Inc.) (Queen and Korn 1984) and compared with published plant DNA sequences (nuclear, mitochondrial and chloroplast sequences). We found that the rice sequence is nearly idential to the ORF39 of the spinach chloroplast DNA, which is downstream from the apocytochrome b\559\ gene (Herrmann et al., 1984). In Fig. 1, the nucleotide and the deduced amino acid sequences for O. rufipogon (O.r) are shown in the center lanes. Only the nucleotides and amino acids that are different from O. rufipogon are indicated for O. sativa IR54 (O.s) sequences above, and those for spinach (Sp) sequences blow, the O. rufipogoti sequences. The DNA sequence includes the last 14 codons of apocytochromc b559, all of ORF39 (nucleotides 1 to 117), and 43 bp of downstream sequences. The ORF39 sequences of the two rice varieties share 98% identical nucleotides (2 substitutions out of 117 nucleotides) and 97% identical amino acids (1 substitution out of 39 amino acids). The OR139 sequences of O. rufipogon and spinach share 92% identical nucleotides (9 differences out of 117) and 92% identical amino acids (3 substitutions out of 39). Immediately after the termination codon of the rice ORF39 sequence, there is a five base pair deletion when compared to the spinach sequence. Beyond that point, the rice and spinach sequences share 84% identity

Fig. 1. Nucleotide and deduced amino acid sequences of ORF39 and its neighboring sequences. Abbreviations used: O.r for Oryza rufipogon, O.s for Oryza sativa IR54, Sp for spinach. Dashes indicate deletions of nucleotides. Underlined sequences are termination codons; TAG for apocytochrome b\559\, and TAA for ORF39.

(7 differences out of 43). In rice and spinach, the ORF39 would code for a 4.4 kDa hydrophobic protein. ORF39 in spinach has been suggested to code for a component of Photosystem II (Herrmann et al. 1984). It is likely that ORF39 codes for a protein because of the high conservation of sequence identity between rice and spinach which are believed to have diverged from each other at least 200 million years ago (Wolfe et al. 1989).

The work presented in this paper was completed in mid 1988, and this paper was written in May 1989. Very recently, Hiratsuka et al. (1989) reported the complete DNA sequence of the rice chloroplast genome O. sativa, cv. Nihonbare, a Japonica variety. The DNA sequence became available to use only in early October. We made a comparison between the sequence of psbL of Nihonbare with the ORF39 sequence of IR54 (an Indica variety) presented in this paper. The two sequences are identical. Thus, ORF39 is in fact psbL.

The rice nucleotide sequences in this article have been submitted to the EMBL/GenBank Data Bank with accession number X15057.

This work was supported by grant RF84066, Allocation No. 3, from the Rockefeller Foundation. J.-C. Cote acknowledges support from Agriculture Canada, Research Station, St.-Jean-sur-Richelieu, Quebec, where he is presently located.

Herrmann, R. G., J. Alt, B. Schiller, W. R. Widger and W. A. Cramer, 1984. Nucleotide sequence of the gene for apocytochrome b-559 on the spinach plastid chromosome: implications for the structure of the membrane protein. FEBS Lett. 176: 239-244.

Hiratsuka, J., H. Shimada, R. Whittier, T. Ishibashi, M. Sakamoto, M. Mori, C. Kondo, Y. Honji, C. R. Sun, B. Y. Meng, Y. Q. Li, A. Kanno, Y. Nishizawa, A. Hirai, K. Shinozaki and M. Sugiura, 1989. The complete sequence of the rice (Oryza sativa) chloroplast genome: Intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Mol. Gen. Genet. 217: 185- 194.

Queen, C. and L. J. Korn, 1984. A comprehensive sequence analysis program for the IBM personal computer. Nucleic Acids Res. 12: 581-599.

Wolfe, K. H., M. Gouy, Y. W. Yang, P. M. Sharp and W. H. Li, 1989. Date of the monocot- dicot divergence estimated from chloroplast DNA sequence data. Proc. Nati. Acad. Sci. USA 86: 6201-6205.

Zhao, X., T. Wu, Y. Xie and R. Wu, 1989. Genome-specific repetitive sequences in the genus Oryza. Theor. Appl. Genet. 78: 201-209.