Bot. Bull. Acad. Sin. (1997) 38: 237-240

Cheng et al. Mung bean mitochondrial lysyl-tRNA gene

The nucleotide sequence of a mung bean (Vigna radiata)

mitochondrial lysyl-tRNA gene

Yuchang Cheng, Yih-Shan Lo, and Hwa Dai1

Institute of Botany, Academia Sinica, Nankang, Taipei, Taiwan 115, Republic of China

(Received April 28, 1997; Accepted July 16, 1997)

Abstract. The nucleotide sequence of a gene coding for tRNALys and its flanking regions from mung bean mitochondrial genome are presented and compared with known mitochondrial tRNALys genes from other higher plant species. This tRNA sequence shows less similarity with its chloroplast counterpart. Several regulatory motifs in the 5' and the 3' flanking regions were identified. The 3' motif could be folded into a stem-loop structure. The functions of these regulatory motifs are discussed.

Keywords: tRNALys; Mitochondria; Mung bean; Vigna radiata.

Abbreviations: DAPI, 4',6-Diamidino-2-phenylindole, hydrochloride.

Introduction

Mitochondrial genomes in plants are large and structurally complex, ranging in size from 200 to 2400 kb (Lonsdale, 1989); however, the plant mitochondrial genomes, unlike their animal counterparts, do not encode the whole set of essential tRNAs required for translation in mitochondria (Hanson and Folkerts, 1992). Systematic investigations of flowering plant mitochondrial tRNAs have revealed that they fall into three groups: the first group includes native tRNAs encoded by the mitochondrial genome; the second group contains chloroplast-like tRNAs with 90_100% homology to their respective chloroplast counterparts; and the final group is cytosolic-like tRNAs that are encoded by nuclear genes (Dietrich et al., 1992; Hanson and Folkerts, 1992). Not all plant species contain the same sets of tRNAs from each of the three different groups. For example, the potato mitochondrial genome encodes 15 tRNAs for the first group, but wheat mitochondrial genome encodes only 10 such tRNAs (Joyce and Gray, 1989; Marechal-Drouard et al., 1990). Hence it is important to determine the number and the origin of tRNAs encoded in the mitochondrial genome of different plant species. Here we report the sequence of a mung bean native mitochondrial tRNALys together with its flanking regions. The sequence is also compared with tRNALys genes of other species.

Materials and Methods

Mitochondrial DNA Extraction

Mitochondria were isolated from 4-day-old etiolated mung bean seedlings (Vigna radiata L. (Wilzed) cv.

Tainan No. 5) by sucrose gradient purification (Dai et al., 1991). After treatment of the purified mitochondria with DNase and protease K, mitochondrial DNA was purified by CsCl-DAPI density gradient (Chiang, 1968).

Cloning of the Mung Bean Mitochondrial tRNALys Gene

Mung bean mitochondrial DNA was digested with EcoRI, and the fragments were cloned into the EcoRI site of the vector pBlueScript II SK(+)(Stratagene). A clone of 3.7 kb, which contained the tRNALys gene, was further subfragmented and recloned into pBlueScript II SK(+). A resultant subclone containing a 2.5 kb PstI-EcoRI fragment was used for sequencing (Figure 1A) (Sambrook et al., 1989).

DNA Sequencing

The sequence analysis was performed as described by the manufacturer on the A.L.F.TM DNA sequencer (Pharmacia).

Results and Discussion

We identified a mung bean mitochondrial gene for tRNALys (trnK(UUU)) located in the internal 2.5 kb PstI-EcoRI fragment of a 3.7 kb EcoRI insert (Figure 1A). The coding sequence of 73 nt (Figure 1B) shows 100% homology with the sequences of mitochondrial tRNAsLys of 3 dicots, common sunflower (Ceci et al., 1996), Arabidopsis (Unseld et al., 1997), rapeseed (Handa and Nakajima, 1992), and 2 monocots, maize (Sangare et al., 1989) and wheat (Joyce and Gray, 1989). However, this coding sequence shows only about 70% similarity when compared with its counterparts in chloroplasts (Sugita et al., 1985; Hiratsuka et al., 1989). Sequence analyses,

1Corresponding author. Fax: (02) 782-7954.


Botanical Bulletin of Academia Sinica, Vol. 38, 1997

Figure 1. A, Restriction map of the 3.7 kb mung bean EcoRI fragment of mitochondrial DNA. The position of tRNALys gene is indicated the arrow indicates the transcriptional direction. B, Nucleotide sequence of the mitochondrial tRNALys gene of mung bean. The region encoding the tRNALys is boxed. The purine-rich sequence, AAGAANRR, is underlined. Direct repeats or inverted repeats are indicated by arrows. The sequence is available from GenBank Caccession number AF013756.

coding region of different species shows a pyrimidine-rich region which is highly conserved among both dicots and monocots (Figure 2). Several short direct repeats could also be identified in this region although it is unknown if these repeats are involved in transcription or processing of tRNA.

When we aligned the 3' downstream from the mung bean tRNALys coding seqence with other species, we found the region is only conserved among dicot plants (Figure 3). Two pairs of possible inverted repeats are present at the 3' end of the mung bean tRNALys coding region, which could form a stem-loop structure within the conserved region (Figures 1B and 3). Although the importance of these stem-loops is not clear, these structures have been implicated in affecting transcription termination and RNA processing of mitochondrial genes (Schuster et al., 1986; Gray et al., 1992)

In summary, we have identified a mung bean native mitochondrial tRNALys gene. Several highly conserved regions were identified in regions upstream and downstream from the tRNALys coding region among different plant species, both dicots and monocots. Further investigation using an in vitro transcription and processing system would be necessary to unravel the functions of these motifs.

therefore, clearly indicate that this 73-nt fragment is a mung bean mitochondrial tRNALys belonging to the first group of tRNA genes. The mung bean tRNALys gene sequence could be folded into the standard cloverleaf secondary structure with no structural deviation (see the figure in Joyce and Gray, 1989). Like other plant mitochondrial tRNA genes, the mung bean mitochondrial tRNALys does not encode the 3'-terminal -CCAOH sequence, which must be added post-transcriptionally (Hanic-Joyce and Gray, 1990).

Analysis of the 5' upstream DNA sequence of the mung bean tRNALys coding region shows a purine-rich motif, which is similar to the 5'-AAGAANRR-3' sequence found between 70 bp and 130 bp upstream of the wheat tRNA coding regions (Figure 1B). Such a motif has also been identified in yeast mitochondrial promoters (Joyce et al., 1988). However, the 5'-NNRAANNNNCRTA-3' promoter motif, which was found immediately upstream from the transcription initiation site of the mitochondrial protein-coding genes, could not be identified within 100 bp upstream of the mung bean tRNALys gene, supporting the notion that different transcription machineries are used for different classes of mitochondrial genes (Muise and Hauswirth, 1992; Rapp and Stern, 1995). Alignment of the 5' upstream sequence of the mitochondrial tRNALys


Cheng et al. Mung bean mitochondrial lysyl-tRNA gene

Figure 2. Alignment of the 5' upstream sequence of the mung bean mitochondrial tRNALys gene with the mitochondrial tRNALys genes of other species. The conserved pyrimidine-rich region is shaded. Numbers refer to nucleotides, starting with -1 at the first nucleotide just 5' from the coding region. Dashes represent gaps introduced for alignment of the respective sequence.

Figure 3. Alignment of the 3' downstream sequence of the mung bean mitochondrial tRNALys gene with mitochondrial tRNALys genes of other species. The conserved region among 3 dicots is shaded. Numbers refer to nucleotides, starting with 74 at the first nucleotide just 3' from the coding region. Dashes represent gaps introduced for alignment of the respective sequence.

Acknowledgements. We are grateful to Dr. K.-S. Chiang for his advice on mitochondrial DNA preparation.

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