34. Insertion of two Tnr1-like transposable DNA elements into the first intron of OsCOX6b1 does not affect transcription of the gene

Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo, 113-8657 Japan

The rice genome has various transposable elements. Many of these elements exist in intergenic spacer regions or introns of various genes and thus contribute to genome diversity. In some cases, insertions of these elements cause specific effects. For example, the insertion of retrotransposon Tos17 into one of the introns of OsABA1 inhibited normal splicing of the gene and greatly reduced the steady state mRNA level of the gene (Agrawal et al. 2001).

Tnr1 is a repetitive sequence in the rice genome. Its copy number was estimated to be about 3500 per haploid genome. This sequence is about 230 bp long, which is too short to encode transposase. However, it has the characteristics of transposable DNA elements; it has terminal inverted repeats of about 75 bp and makes target sequence duplications upon transition. Tnr1 has some other unique features, including transposition to the specific sequence 5'-PuTAPy-3', duplicating the TA sequence. Therefore, Tnr1 is thought to be a kind of transposable DNA element, and it is probably non-autonomous (Umeda et al 1991; Tenzen et al. 1994).

We previously characterized a cDNA of a novel rice nuclear-encoded gene for the subunit VIb of cytochrome c oxidase (COX) (Ohtsu et al. 1999). COX is the terminal oxidase of the cytochrome electron transport pathway that produces ATP in mitochondria. The deduced amino acid sequence of this gene, designated OsCOX6b1, has an extended N-terminal sequence compared with the human (Taanman et al. 1989) and yeast (LaMarche et al. 1992) COX6bs. We subsequently determined the genomic nucleotide sequence of OsCOX6b1.

First, we obtained genomic DNA fragments containing the whole OsCOX6b1 coding region by PCR using rice total DNA as a template. The nucleotide sequence of the fragments was completely determined (accession number: AB047975). Based on the OsCOX6b1 cDNA sequence, the OsCOX6b1 gene contained four introns. As shown in Figure 1A, we found two Tnr1-like transposable DNA elements in the first intron. The upstream and downstream Tnr1-like elements had 56% and 60% identities to Tnr1A (previously identified Tnr1), respectively. Arabidopsis has a similar gene (designated AtCOX6b1). The nucleotide sequence of this gene (determined from Arabidopsis EST clone E10B10) was found to have four introns at the same positions as OsCOX6b1, and its deduced amino acid sequence has a long N-terminal sequence like OsCOX61. However, no transposable elements such as Tnr1 were found in the introns of AtCOX6b1 (Fig. 1A).

To determine the effect of Tnr1 insertion into the first intron of OsCOX6b1, we investigated the steady-state mRNA levels of both OsCOX6b1 and AtCOX6b1 in various organs. As shown in Figure 1B, the steady-state level of OsCOX6b1 mRNA, as well as that of AtCOX6b1, was detected in the mature form in all organs examined in this report. Based on the sizes of the transcripts, the first introns of both OsCOX6b1 and AtCOX6b1 were normally spliced out at the same positions. Based on the results obtained with AtCOX6b1, the amounts of the steady-state

mRNA of OsCOX6b1 appeared to be normal. These results indicate that the insertion of Tnr1-like transposable DNA elements into the first intron of OsCOX6b1, unlike the insertion of Tos17 into one of the introns of OsABA1, does not affect the splicing position of the intron, and likely does not affect the steady-state mRNA level of the gene.


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