Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol158.Issue1-2.Sept2000:

Identification and characterization of the

trnS

/

pseudo-tRNA

/

nad

3

/

rps

12 gene cluster from

Coix lacryma

-

jobi

L: organization, transcription and RNA editing

Sandra Martha G. Dias

a

_{, Susely F. Siqueira}

a

_{, Bernard Lejeune}

b

_{, Anete P. de Souza}

a,c,

_*

a_{Centro de Biologia Molecular e Engenharia Gene´tica(CBMEG),Uni6ersidade Estadual de Campinas(UNICAMP),} Cidade Uni6ersita´ria‘Zeferino Vaz’,13083-970C.P.6010Campinas SP,Brazil

b_{Institut de Biotechnologie des Plantes,Uni6ersite´ de Paris Sud,Baˆtaille630,91405Orsay Cedex,France} c_{Departamento de Gene´tica e E6oluc¸a˜o,Instituto de Biologia(IB),Uni6ersidade Estadual de Campinas(UNICAMP),}

Cidade Uni6ersita´ria‘Zeferino Vaz’,13083-970C.P.6109Campinas SP,Brazil

Received 13 April 2000; received in revised form 31 May 2000; accepted 2 June 2000

Abstract

During a study of mitochondrial sequence conservation between the liverwortMarchantia polymorphaand several Angiosperm species, as revealed by heterologous hybridization experiments, thetrnS/pseudo-tRNA/nad3/rps12 gene cluster inCoix lacryma

-jobi L., an Asian grass species from the Andropogoneae, was identified using the mitochondrial probe orf167 from M.

polymorpha. TheCoix gene cluster was cloned and sequenced, and its expression analyzed. The gene sequence and gene locus organization were found to be similar to the corresponding cluster in wheat and maize. Northern hybridization and reverse transcription-polymerase chain reaction analyses indicated that nad3 and rps12 genes were co-transcribed as a 1.25 kb RNA molecule. The transcript displayed 20 and six RNA edition sites, in thenad3 andrps12 genes, respectively, that changed the codon identities to amino acids, which are better conserved in different organisms. Twenty-three cDNA clones were analysed for the edition process and revealed different partial editing patterns without apparent sequential processing. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords:Mitochondrial DNA; NADH dehydrogenase subunit 3; Ribosomal S12; RNA editing;Coix lacryma-jobiL.

www.elsevier.com/locate/plantsci

1. Introduction

The mitochondrial genomes of higher plants are much larger than those of non-plant organ-isms, from which they differ in general structure, variable gene arrangement, encoding capacity, and gene expression [1,2]. Almost all of the genes for protein complexes in the respiratory

chain, encoded by animal mitochondrial

genomes, have also been identified in higher plant mitochondria. The mitochondrial genome of Arabidopsis thaliana contains 57 genes with at least 42 putative open reading frames [3]. RNA editing occurs widely in higher plant mitochon-dria and involves C to U and, less frequently, U to C alterations [4 – 6].

General understanding of the information con-tent of plant mitochondrial genomes has been greatly advanced by the sequencing of the entire

mitochondrial genome of the liverwort

Marchan-tia polymorpha [7] and of the higher plant A. thaliana [3]. In the M. polymorpha mitochondrial

genome, 28 open reading frames (orf ) were

pre-dicted as being possible genes. Five of these (orf 228, 509, 169, 322 and 277) are homologous to

* Corresponding author. Present address: Centro de Biologia Molecular e Engenharia Gene´tica (CBMEG), Universidade Estadual de Campinas (UNICAMP), Cidade Universita´ria ‘Zeferino Vaz’, 13083-970 C.P. 6010 Campinas SP, Brazil. Tel.: +55-19-7881132; fax: +55-19-7881089.

E-mail address:anete@unicamp.br (A.P. de Souza).

0168-9452/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 0 ) 0 0 3 0 8 - 3

genes required for cytochrome c biogenesis in Rhodobacter capsulatus, a photosynthetic bac-terium. Homologous genes to those already men-tioned have also been found in higher plant species [8 – 16].

Genes are widely dispersed in the mitochondrial genome of higher plants and their disposition varies considerably among plant species. This vari-ability is the result of frequent rearrangements within mitochondrial genomes [2]. Co-transcrip-tion of adjacent genes has been found to occur in the mtDNA of several plant species. Polycistronic

transcripts such as the rrn18/rrn5 gene in wheat

[17], and rps3/rpl16 [18] and atpA/atp9 genes in

maize [19] may contain sequence coding for re-lated products. However, co-transcribed genes

such as atp9/rps13 in tobacco [20], nad3/rps12 in

wheat and maize [21], and orf25/cox3 in rice [22]

are involved in different metabolic pathways. The co-transcription of genes encoding proteins, acting in different metabolic pathways, indicates that

post-transcriptional and/or translational

regula-tion is important for the control of gene-product abundance [16].

Ribosomal protein genes are generally scattered throughout the mitochondrial genome of An-giosperms and, in some cases, may be linked to non-ribosomal protein genes [8,9,16,21]. Some of these associations have been conserved over large

evolutionary distances. For example, the nad3

gene (encoding mitochondrial

NADH-ubiquinone-oxidoreductase subunit 3) and the rps12 gene

(small subunit ribosomal protein 12) are closely linked and co-transcribed in the mtDNA of An-giosperm families as distant as the monocot

grasses (Sorghum [23], rice [24], maize and wheat

[21]) and the dicot Brassicaceae (Arabidopsis [25],

radish [26], rapeseed and other Brassica species

[16]) and also in the mtDNA fromPinus syl6estris

and other Gymnosperms [27]. In maize and wheat,

the trnS and a pseudo-tRNA gene are located

upstream to nad3 [21].

In this report, the analysis of a 1.4 kb BglII

fragment from the mtDNA of Coix lacryma-jobi

L. (a south-east Asia grass species from the An-dropogoneae tribe) identified by heterologous

hy-bridization with DNA sequence of the

mitochondrial orf167 of M. polymorpha, was

de-scribed. This orf I67 was found to hybridize with

C. lacryma-jobi nad3 gene, located on a gene

cluster that includestrnS, pseudo-tRNA andrps12

genes. The organization, sequence analysis, tran-scription and editing pattern of this gene cluster are described.

2. Materials and methods

2.1. Isolation and analysis of mtDNA and mtRNA Mitochondrial DNA (mtDNA) was isolated

from etiolated seedlings of C. lacryma-jobi L. cv

Adlay, maize (Zea mays, cv AGF352), pea (Pisum sati6_{um, cv Mikado), soybean (Glycine max, cv}

IAC-5) and alfalfa (Medicago sati6um, from a

local market), potato tubers (Solanum tuberosum, cv Binje) and cauliflower inflorescence (Brassica oleracea, from a local market). Purified mitochon-dria were prepared as previously described [28]: mtDNA was obtained after mitochondrial lysis and CsCl-ethidium bromide centrifugation. Mito-chondrial RNA was prepared as described previ-ously [29], treated with DNAse I, to remove the remaining DNA, then phenol extracted and pre-cipitated. Standard procedures [30] were used for restriction enzyme digestions and agarose gel electrophoresis.

2.2. Southern and Northern hybridizations

Restriction-digested mtDNAs were transferred to Hybond-N filters (Amersham, UK) by standard procedures [30]. DNA fragments used as probes were purified from gel slices by electroelution and

labeled by random hexamer priming.

Het-erologous hybridizations, using M. polymorpha

mitochondrial orf167 as a probe against Bam

HI-digested mtDNA from different Angiosperm

spe-cies, were performed under low stringency

conditions: the filters were pre-hybridized (45°C, 4

h) and hybridized (45°C, 18 h) in 5×SSC, 10×

Denhardt’s solution, 0.1% (w/v) sodium dodecyl

sulfate (SDS), 20 mM Tris – HCl (pH 7.5) and 0.1

mg/ml denatured salmon-sperm DNA, plus a

probe which had been32_{P-labeled by a multiprime}

reaction (Prime-a-Gene kit; Promega). Filters were

washed twice for 15 min in 1×SSC, 0.1% SDS

solution at 50°C and then exposed for autoradiog-raphy. Homologous hybridizations and washes were performed under stringent conditions: pre-hybridization (42°C, 4 h) and pre-hybridization (42°C, overnight) were carried out in a solution

contain-ing 5×SSC, 10×Denhardt’s, 1.0% (w/s) SDS, 20

mM Tris – HCl (pH 7.5), 0.1 mg/ml denatured

salmon-sperm and 50% formamide. The filter was

then washed twice for 10 min in 2×SSC, 1% SDS

at room temperature, and twice for 10 min with

0.1×SSC, 0.1% SDS at 42°C, and then exposed

for autoradiography. Isolated mtRNA (5.0 mg/

lane) was denatured at 55°C for 15 min in a solution containing 18% formaldehyde, 50%

for-mamide and 0.5×MOPS buffer. The samples

were fractionated on 1.2% agarose gel containing

1×MOPS buffer and 0.66 M formaldehyde and

blotted onto Hybond N. New membrane strips were used for each hybridization experiment. Pre-hybridization and Pre-hybridization were performed under stringent conditions. Probe labeling, hy-bridization and washes were as already described. Transcript sizes were determined using RNA size standards from Gibco-BRL (USA).

2.3. cDNA synthesis and polymerase chain reaction amplification

Reverse transcription was carried out on 1.0 mg

DNAseI-treated mtRNA using 10 pmol from an appropriate primer. After heating the mixture for 10 min at 65°C, the buffer from an ‘Expanded™ Reverse Transcriptase’ kit (Boehringer, Germany), 10 mM dithiothreitol, 1 mM of each dNTP, 20 U RNAsine (Gibco-BRL) and 50 U Expand Reverse Transcriptase was added. The reaction was incu-bated for 1 h at 42°C and stopped by heating for 2 min at 95°C. The resulting cDNAs were am-plified by polymerase chain reaction (PCR) using 10 pmol of the appropriate primers in a buffer supplied by the manufacturer, 0.15 mM of each

dNTP, 2.5 mM MgCl2 and 2.5 U Taq DNA

Polymerase (Gibco BRL) in a final reaction

vol-ume of 100ml. The annealing temperature used for

the primer combinations fnad3 – drps12 and

dnad3 – drps12 was 61°C. After 3 min at 94°C, 25 cycles of amplification were carried out (94°C for 1.5 min, 61°C for 2 min, 72°C for 2 min), followed by a final extension of 10 min at 72°C. The amplified products were purified by agarose gel electrophoresis before cloning. Control for cDNA synthesis was performed replacing reverse tran-scriptase by water in the reaction mixtures and verifying through PCR that there was no amplifi-cation product from that template.

2.4. cDNA cloning and sequencing

Standard procedures were used in the prepara-tion, isolation and analysis of recombinant clones of Escherichia coli [30]. Overlapping DNA restric-tion fragments from the region of interest were cloned into pBluescript vectors (Stratagene, USA) and the cDNAs generated by reverse transcriptase-polymerase chain reaction (RT-PCR) were cloned into the pGEM-T vector System I (Promega, USA). Nucleotide sequences were determined us-ing the dye terminator-cycle method on an ABI

PRISM 310 sequencer (Applied Biosystems,

USA). Both strands of the cDNA and genomic clones were sequenced. The sequence data was analyzed using the Lasergene System (DNA Star, USA).

2.5. PCR amplification of orf167 from M. polymorpha mtDNA

M. polymorpha total DNA (10 – 20 ng) (kindly provided by M.-C. Boisselier-Dubayle, Museum National d’Histoire Naturelle, Laboratoire de Cryptogamie, Paris, France) were mixed with 2.5

mM MgCl2 on a buffer supplied by the

manufac-turer, containing 0.1 mM of each dNTP and 2.5 U

Taq DNA Polymerase (Gibco BRL) in a final

reaction volume of 100ml. After 3 min at 94°C, 30

cycles of amplification were carried out (92°C for 1 min, 55°C for 1.5 min, 72°C for 2 min), followed by a final extension of 10 min at 72°C. The products of amplification were cloned into the pGEM-T vector and sequenced to confirm their identity before using them as probes in het-erologous hybridizations.

2.6. Oligonucleotides

For PCR amplification of theorf167 from total

DNA of the M. polymorpha, the primers used

were: marpo 167a, 5%_{-AGT TGG AGG AGA}

TAG GAT TTC GTG T-3%; and marpo167b, 5%

-GTT ACT TCT TTT GCG GCT -GTT TTC T-3%_.

For cDNA synthesis and RT-PCR amplification,

the primers were: fnad3, 5%_{-GCG AGA GAA}

CGA AGT GGG-3%; dnad3, 5%-GCT TTG GTG

ATG TCG GAA T-3%; and drps12, 5%-GAG GCA

3. Results and discussion

3.1. Isolation of the trnS/pseudo-tRNA/nad3/rps12 locus from Coix mt DNA

Several reports have shown that M. polymorpha

mitochondrial orfs, with initially unknown

func-tions, have been conserved in higher plants. The

identity of these orfs was discovered by

compari-sons with sequence data banks [8 – 16]. To identify M. polymorpha mitochondrial orfs conserved in higher plants, the extent of mitochondrial

se-quence conservation between M. polymorpha and

Angiosperms were assessed. Several mitochondrial

orfs from the liverwort have been used as probes

against mtDNA from maize, Coix, cauliflower,

potato, pea and soybean in heterologous hy-bridization experiments. Appropriate oligonucle-otides were designed to amplify some chosen

mitochondrial orfs from M. polymorpha total

DNA. The experimental hybridization conditions were chosen to identify mtDNA sequences show-ing as little as 50% sequence similarity with the

probe together with keeping a good signal/noise

ratio. One of the probes, namely orf167,

hy-bridized with only one or two restriction frag-ments from each of the species listed giving a

stronger signal with maize andCoixmitochondrial

DNAs (Fig. 1). Therefore, orf 167 from M.

poly-morpha could be considered a potentially

con-served orf in Angiosperms. The hybridizing

sequence inCoixmt was located on aBglII 1.4 kb

fragment (data not shown) that was cloned into a pBluescript and characterized by restriction

map-ping (Fig. 2). The orf167 similarity region was

localized on the restriction map of this BglII

frag-ment by hybridization (results not shown).

3.2. Sequence analysis of the

trnS/pseudo-tRNA/nad3/rps12 locus fn Coix mtDNA

The 1.4 kb BglII fragment was subcloned into

pBluescript to obtain appropriate overlapping fragments for DNA sequencing on both strands. The complete nucleotide sequence of the fragment (Fig. 2) was compared with DNA sequence data-bases. The comparisons identified significant

simi-larities between the 1.4 kb BglII sequence and a

cluster of mitochondrial genes of wheat and maize,

encoding the following genes: tRNASer _(trnS),

pseudo-tRNA, subunit 3 of NADH dehydroge-nase (nad3) and ribosomal protein subunit S12

(rps12) [21]. Since the sequence of this rps12 gene

was interrupted by one of the terminal BglII sites

of the fragment, primer drps12 (rps12 maize-wheat sequence based) in combination with fnad3 primer was used to amplify the missing portion of the

Fig. 1. Identification of mtDNA restriction fragments con-taining sequences homologous to M. polymorpha mitochon-drialorf167. Mitochondrial DNA from alfalfa (1), potato (2),

Coix (4), cauliflower (5), pea (6), maize (7), soybean (8) and

Coix ctDNA (3) was digested with BamHI and fractionated on 1% agarose gel. The DNA was then transferred to a nylon filter and hybridized with a 32_{P-labeled fragment containing}

orf167. M, Molecular markerl DNA digested withHindIII andfX 174 DNA digested withHaeIII.

Fig. 2. Physical map of the restriction sites of theCoix1.4 kb

BglII/BglII fragment. Genes are represented by dark boxes. The four probes used in Northern and Southern blotting are indicated and correspond tonad3 (probe 1),rps12 (probe 2), tRNASer _{(probe 3) and pseudo-tRNA (probe 4). The arrows}

indicate gene orientation. The restriction sites are: A, A6aI; Bg,BglII; E,EcoRI; Ps,PstI; Pv,P6_{uII; S,SstI; S,SpeI; X,}

XhoI. ThePstI andSstI sites are from the BlueScript plasmid vector, from which 1.4BglII/BglII was cloned.

Fig. 3. Nucleotide sequence of the tRNASer_{/pseudo-tRNA/nad3/rps12 locus in Coix mtDNA (GenBank accession number}

AF239263). The amino acid sequence deduced from the nucleotide sequence is shown in single letter code above the nucleotide sequence. RNA editing sites are indicated by a lowercase ‘c’. The predicted amino acid before and after editing are indicated. Horizontal arrows, synthetic oligonucleotides prepared for cDNA synthesis and PCR amplification; dashed line, pseudo-tRNA insertion; stars, stop codons. Pseudo-tRNA and tRNASer _{are underlined. The sequence used for drps12 synthesis was based on}

a consensus sequence between maize and wheat [21].

rps12 gene from Coix mtDNA. The amplification product was cloned and sequenced, and its

se-quence added to that of the 1.4 kbBglII fragment

(Fig. 3).

The stringency conditions for heterologous

hy-bridizations usingorf167 were chosen for detecting

low similarity with mtDNA sequences. Sequence comparisons and alignment between the sequences

of orf167 and the BglII fragment revealed that

orf167 displayed 45% similarity with theCoix nad3

gene; this similarity was evenly distributed over the gene sequence and responsible for the hybridization signal as checked by hybridization controls with the

nad3 sequence from wheat (data not shown). This value is to be compared with the more significant 82% similarity found when comparing this same Coix nad3 gene with the actual homologous nad3 from M. polymorpha. Although orf167 has been described as a hypothetical 18.6 KD protein in the nad3 –nad7 intergenic region, with no relationship to the NAD3 protein, the origin of the similarity with nad3 reported here is intriguing. It may be fortuitous or it may reveal an ancient sequence

duplication in the mitochondrial genome of M.

polymorpha having occurred in the vicinity of the

The nucleotide sequences of the Coix nad3/ rps12 gene show 100 – 99.4 and 100 – 99.7% simi-larity with the same gene sequences from maize

and wheat, respectively. In Coix, the intergenic

spacer sequence between nad3 and rps12 is 44 bp

long, very similar to the 47 bp intergenic spacer found in maize and wheat [21].

The Coix tRNASer _{sequence has 100% identity}

with the same gene from maize, wheat and rice [21,24,31]. There is a pseudo-tRNA sequence

lo-cated 259 bp downstream of the trnS gene,

ho-mologous to the sequence found in maize, wheat and rice mitochondrial genomes [21,24,31]. The

Coix pseudo-tRNA gene presents a 50 bp

inser-tional sequence in the same position as in other monocots examined so far (Fig. 3), which is 99% similar to a 48 bp insertion found in the maize pseudo-tRNA. As pointed out previously [24], the rice pseudo-tRNA gene seems to have evolved

from tRNAPhe _{because of its high sequence}

ho-mology with tRNAPhe _{[32]. Sequence comparisons}

excluding insertional sequences have shown that

Coixand maize pseudo-tRNA genes are 93%

sim-ilar with the potato tRNAPhe_{. Thus, in all}

mono-cots examined so far [21,24,31], this pseudo-tRNA gene seems to have been inactivated in the course of evolution. This inactivation may have origi-nated from an intramolecular recombination fol-lowed by a sequence insertion in a common ancestor sequence, probably the mitochondrial

tRNAPhe _gene.

Southern blot hybridizations using thenad3 and

pseudo-tRNA genes as probes against Coix,

maize, cauliflower, potato, soybean, pea and

al-falfa mitochondrial genomes digested by BamHI,

showed that the nad3 gene is present as a single

copy in mtDNA from Coix and other species

(data not shown). However, of the presented

spe-cies, only the maize and Coix mtDNAs gave

strong hybridization signals with pseudo-tRNA gene (data not shown). These results suggest that this pseudo-tRNA gene originated from a recom-bination event that took place in a monocot

an-cestor, after the dicot/monocot divergence.

3.3. Transcription of the

trnS/pseudo-tRNA/nad3/rps12 locus

Northern blot hybridizations were carried out

with total mitochondrial RNA from Coix using

probes corresponding to each of the genes in the

cluster: a 0.5 kb P6_{uII/SpeI fragment (probe 1)}

for nad3, a 0.3 kb SstI/XhoI fragment (probe 2)

for rps 12, and a 0.6 P6_{uII/PstI fragment (probe}

3) for trnS (Fig. 2). Only a single abundant

tran-script of approximately 1250 nts was detected

with probes 1 and 2, indicating that the Coix nad3

and rps12 genes were co-transcribed, as confirmed by RT-PCR amplification (see later). This

tran-script pattern was similar to that of nad3/rps12

transcription in rice, where a single 1.2 kb tran-script was identified [24]. However, maize and wheat have shown a more complex transcription pattern, with two and at least four transcripts, respectively [21], and the mRNA is 0.9 kb in length. The mRNA transcript size difference

be-tween Coix and maize was surprising, considering

the high sequence identity (95 – 98%) shared

be-tween the 1455 pb Coix trnS/pseudo-tRNA/nad3/

rps12 locus and the same region in maize and

wheat. Considering that the two mapped 5% _ends

of the wheat transcript [21] are within a sequence region showing almost 100% identity with the

Coix sequence, it can be supposed that the

differ-ence observed in transcript size would be

ex-plained by the 3%_{end transcript change. However,}

ends of the nad3/rps12 Coixtranscript need to be

mapped to confirm this hypothesis.

Northern hybridizations were used to test

whether the trnS gene and pseudo-tRNA were

transcribed in Coix. In this way, probes 3 and 4

(Fig. 2) were hybridized with Coix total mtRNA.

Fig. 4. RNA hybridizations of thenad3, rps12 and tRNASer

transcripts in Coix. Five micrograms of total mtRNA were denatured and applied to each lane of a 1.2% agarose/ formal-dehyde gel, transferred to a nylon membrane and hybridized

32_{P-labeled fragments ofnad3 (1),rps12 (2) andtRNA}Ser_(3).

An RNA ladder (9.5 – 0.24 kb, BRL) was used to estimate molecular size.

Table 1

Degree of editing of the nad3 andrps12 genes in the 23 cDNA samples analyzed

nad3

Editing rps12

CDNA Number of edited sites

CDNA Number of edited sites

clones clones

% N

N %

26 20

Completely edited 6 12 53 6

Incompletely but extensively edited 10 43 18–19 4 17 5

Barely edited 2 9 1 3 13 2

22 0 4

5 17

Completely unedited 0

23

Total 23

Only probe 3 hybridized to a transcript (Fig. 4),

indicating that trnSis transcribed inCoixwhereas

the pseudo-tRNA is not.

As already mentioned, nad3-rps12

co-transcrip-tion was confirmed by RT-PCR amplificaco-transcrip-tion. Three primers (fnad3, dnad3 and drps12) were

designed from the coding regions of the nad3 and

rps12 genes (Fig. 3). An approximately 800 bp

amplification product was obtained for the nad3/

rps12 cotranscript.

3.4. RNA editing pattern of the nad3/rps12 transcripts in Coix

To characterize the extent and sites of RNA

editing in nad3/rps12 transcripts, cDNA covering

the coding region was obtained by RT-PCR using the primers fnad3 or dnad3 and drps12 (Fig. 3). The comparison of the genomic and 23 cDNA

revealed 20 C-to-U RNA editing events in nad3

and six in therps12 coding region (Fig. 3).

Twenty-six C-to-U editing sites resulted in 16 and Twenty-six codon

modifications in thenad3 and rps12 genes,

respec-tively, corresponding to 13.6% of the NAD3 and 4.8% of the RPS12 amino-acid sequence. Most of

the editing sites in theCoix nad3 –rps12 genes have

already been found in wheat [21], Oenothera [33],

Pinus [27], Magnolia, onion, sunflower [34],

Petu-nia [35], Sorghum [23] and Brassica [16]. A

com-parison of the nad3 editing positions in Coix and

wheat [36] showed four differences: codons 13, 42 and 46 were edited only in wheat, and codon 62

(editing site six in Fig. 3) only inCoix, whereas the

rps12 editing sites in Coix and wheat were identi-cal. Contrary to the case with wheat, these four

editing events inCoix nad3 were identical to those

inOenothera[33] and only one of them was

differ-ent inPinus[27]. These sites probably existed prior

to the evolutionary separation of monocots and dicots, and the species-specific differences origi-nated from genomic point mutations in some lin-eages during evolution.

Sequence analysis of 23 cDNA clones revealed unedited and partially edited clones with no evi-dent polarity for the editing process. The extent of

editing differed between nad3 and rps12

tran-scripts: more than 50% of them presented

com-pletely edited rps12, whereas only 26% of them

were fully edited in thenad3 region (Table 1). This

finding suggests that the rps12 editing sites inCoix

are edited first or faster than thenad3 editing sites.

This pattern differs from that in Magnolia where

all 23 editing sites in the PCR-derivednad3 cDNA

were found to be fully edited, while all of the

editing sites in rps12 were only partially altered

[34]. Mixtures of partially or differentially edited

cDNA clone populations derived from the nad3 –

rps12 loci have also been found in the mitochon-dria of other plants such as wheat [36] and pine [27].

In conclusion, a high conservation of the trnS/

pseudo-tRNA/nad3/rps12 sequence, gene

organi-zation and editing pattern of thenad3/rps12 genes

among Coix and monocots were found, especially

between maize and Coix, indicating that evolution

has produced a high degree of conservation in this locus in these species. This fact is surprising

consid-ering; first, Coix is a distant relative of maize (an

Asian species of the Andropogoneae tribe); second, the high recombinationary ability of the mitochon-drial genome plant; and, finally, the general non-conservation of different gene clusters, even in very close evolutionarily species.

Acknowledgements

The authors wish to thank Dr. M.-C. Boisselier-Dubayle (Museum National d’Histoire Naturelle, Laboratoire de Cryptogamie, Paris, France) for

the gift of Marchantia polymorpha total DNA.

This work was financed by grants to A.P.S. from Fundac¸a˜o de Amparo a` Pesquisa de Sa˜o Paulo

(FAPESP; 96/03520-8). A.P.S. was also the

recipi-ent of a research fellowship from Conselho

Na-cional de Desenvolvimento Cientı´fico e

Tecnolo´gico (CNPq). S.M.G.D. and S.F.S. were supported by graduate fellowships from FAPESP.

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[11] G. Bonnard, J.M. Grienenberger, A gene proposed to encode a transmembrane domain of an ABC transporter is expressed in wheat mitochondria, Mol. Gen. Genet. 246 (1995) 91 – 99.

[12] W. Jekabson, W. Schuster, Orf250 encodes a second subunit of an ABC-type transporter inOenothera mito-chondria, Mol. Gen. Genet. 246 (1995) 166 – 173. [13] M. Nakazono, Y. Ito, N. Tsutsumi, A. Hirai, The gene

for a subunit of an ABC-type heme transporter is tran-scribed together with the gene for subunit 6 of NADH dehydrogenase in rice mitochondria, Curr. Genet. 29 (1996) 412 – 416.

[14] H. Handa, G. Bonnard, J.M. Grienenberger, The rape-seed mitochondrial gene encoding a homologue of the bacterial proteinCcl1 is divided into two independently transcribed reading frames, Mol. Gen. Genet. 252 (1996) 292 – 302.

[15] R. Menassa, N. El-Rouby, G.G. Brown, An open read-ing frame for a protein involved in cytochromec biogen-esis split into two parts inBrassicamitochondria, Curr. Genet. 31 (1997) 70 – 79.

[16] K. Itani, H. Handa, Rapeseed mitochondrial ccb206, a gene involved in cytochrome c biogenesis, is co-tran-scribed with the nad3 and rps12 genes: organization, transcription, and RNA editing of the nad3/rps12/

ccb206 locus, Curr. Genet. 34 (1998) 318 – 325.

[17] D. Falconet, B. Lejeune, F. Quetier, M.W. Gray, Evi-dence for homologous recombination between repeated sequences containing 18S and 5S ribosomal RNA genes in wheat mitochondrial DNA, EMBO J. 3 (1984) 297 – 302.

[18] M.D. Hunt, K.J. Newton, The NC3 mutation-genetic-evidence for the expression of ribosomal-protein genes in

Zea maysmitochondria, EMBO J. 10 (1991) 1045 – 1052. [19] P.S. Covello, M.W. Gray, Sequence-analysis of wheat mitochondrial transcripts cappedin6_{itro: definitive}

iden-tification of transcription initiation sites, Curr. Genet. 20 (1991) 245 – 251.

[20] M.M. Bland, C.S. Leavings III, D.F. Matzinger, The tobacco mitochondrial ATPase subunit 9 gene is closely linked to an open reading frame for a ribosomal protein, Mol. Gen. Gen. 204 (1986) 4 – 8.

[21] J.M. Gualberto, H. Wintz, J.H. Weil, J.M. Grienen-berger, The genes coding for subunit 3 of NADH dehy-drogenase and for ribosomal protein S12 are present in the wheat and maize mitochondria genomes and are co-transcribed, Mol. Gen. Genet. 215 (1988) 118 – 127. [22] A.W. Liu, K.K. Narayanan, C.P. Andre´, E.K. Kaleikau,

V. Walbot, Co-transcription oforf25 andcoxIIIin rice mitochondria, Curr. Genet. 21 (1992) 507 – 513. [23] W. Howad, F. Kempken, Sequence analysis and

tran-script processing of the mitochondrialnad3 –rps12 genes from Sorghum bicolor, Plant Sci. 129 (1997) 65 – 68. [24] T. Suzuki, S. Kazama, A. Hirai, T. Akihama, K.

Kad-owaki, The rice mitochondrial nad3 gene has an ex-tended reading frame at its 5%_{end: nucleotide sequence}

analysis of ricetrnS,nad3, andrps12 genes, Curr. Genet. 20 (1991) 331 – 337.

[25] P. Brandt, S. Su¨nkel, M. Unseld, A. Brennicke, V. Knoop, The nad4L gene is encoded between exon c of

nad5 and orf25 in the Arabdopsis mitochondrial genome, Mol. Gen. Genet. 236 (1992) 33 – 38.

[26] C.A. Makaroff, J.D. Palmer, Mitochondrial DNA rear-rangements and transcriptional alterations in the male sterile cytoplasm of Ogura radish, Mol. Cell Biol. 8 (1988) 1474 – 1480.

[27] B. Karpinska, S. Karpinski, J.E. Hallgren, The genes encoding subunit 3 of NADH deydrogenase and riboso-mal protein S12 are co-transcribed and edited inPinus syl6estris (L.) mitochondria, Curr. Genet. 28 (1995) 423 – 428.

[28] F. Vedel, F. Quetier, Physico-chemical characterization of mitochondrial DNA from potato tubers, Biochim. Biophys. Acta 340 (1974) 374 – 387.

[29] D.B. Stern, K.J. Newton, Isolation of plant mitochon-drial RNA, Methods Enzymol. 118 (1986) 488 – 496. [30] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular

cloning: a laboratory manual, 2nd ed. (1989), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [31] H. Wintz, J.M. Grienenberger, J.H. Weil, D.M.

Lons-dale, Location and nucleotide sequence of two tRNA genes and a tRNA pseudogene in maize mitochondrial genome: evidence for the transcription of a chloroplast gene in mitochondria, Curr. Genet. 13 (1988) 247 – 254. [32] L. Mare´chal, P. Guillemault, J.M. Grienenberger, G. Jeannin, J.H. Weil, Structure of bean mitochondrial

tRNAPhe _{and localisation of the tRNA}Phe _{on the}

mito-chondrial genomes of maize and wheat, FEBS Lett. 184 (1985) 289 – 293.

[33] W. Schuster, B. Wissinger, M. Unseld, A. Brennicke, Transcripts of the NADH-dehydrogenase subunit 3 gene are differentially edited in Oenothera mitochondria, EMBO J. 9 (1990) 263 – 269.

[34] G. Perrotta, T.M. Regina, L.R. Ceci, C. Quagliariello, Conservation of the organization of the mitochondrial

nad3 and rps12 genes in evolutionary distant an-giosperms, Mol. Gen. Genet. 251 (1996) 326 – 337. [35] B. Lu, M.R. Hanson, A single nuclear gene specifies the

abundance and extent of RNA editing of a plant mito-chondrial transcript, Nucleic Acids Res. 20 (1992) 5699 – 5703.

[36] J.M. Gualberto, G. Bonnard, L. Lamattina, J.M. Grienenberger, Expression of the wheat mitochondrial

nad3 –rps12 transcription unit: correlation between edit-ing and mRNA maturation, Plant Cell 3 (1991) 1109 – 1120.

3. Results and discussion

3.1. Isolation of the trnS/pseudo-tRNA/nad3/rps12

locus from Coix mt DNA

Several reports have shown that M. polymorpha

mitochondrial orfs, with initially unknown

func-tions, have been conserved in higher plants. The identity of these orfs was discovered by compari-sons with sequence data banks [8 – 16]. To identify

M. polymorpha mitochondrial orfs conserved in higher plants, the extent of mitochondrial se-quence conservation between M. polymorpha and Angiosperms were assessed. Several mitochondrial

orfs from the liverwort have been used as probes

against mtDNA from maize, Coix, cauliflower,

potato, pea and soybean in heterologous hy-bridization experiments. Appropriate oligonucle-otides were designed to amplify some chosen

mitochondrial orfs from M. polymorpha total

DNA. The experimental hybridization conditions were chosen to identify mtDNA sequences show-ing as little as 50% sequence similarity with the probe together with keeping a good signal/noise

ratio. One of the probes, namely orf167,

hy-bridized with only one or two restriction frag-ments from each of the species listed giving a stronger signal with maize andCoixmitochondrial DNAs (Fig. 1). Therefore, orf 167 from M. poly

-morpha could be considered a potentially

con-served orf in Angiosperms. The hybridizing

sequence inCoixmt was located on aBglII 1.4 kb fragment (data not shown) that was cloned into a pBluescript and characterized by restriction map-ping (Fig. 2). The orf167 similarity region was localized on the restriction map of this BglII frag-ment by hybridization (results not shown).

3.2. Sequence analysis of the

trnS/pseudo-tRNA/nad3/rps12 locus fn Coix mtDNA

The 1.4 kb BglII fragment was subcloned into pBluescript to obtain appropriate overlapping fragments for DNA sequencing on both strands. The complete nucleotide sequence of the fragment (Fig. 2) was compared with DNA sequence data-bases. The comparisons identified significant simi-larities between the 1.4 kb BglII sequence and a cluster of mitochondrial genes of wheat and maize, encoding the following genes: tRNASer _(trnS),

pseudo-tRNA, subunit 3 of NADH dehydroge-nase (nad3) and ribosomal protein subunit S12 (rps12) [21]. Since the sequence of this rps12 gene was interrupted by one of the terminal BglII sites of the fragment, primer drps12 (rps12 maize-wheat sequence based) in combination with fnad3 primer was used to amplify the missing portion of the

Fig. 1. Identification of mtDNA restriction fragments con-taining sequences homologous to M. polymorpha mitochon-drialorf167. Mitochondrial DNA from alfalfa (1), potato (2), Coix (4), cauliflower (5), pea (6), maize (7), soybean (8) and Coix ctDNA (3) was digested with BamHI and fractionated on 1% agarose gel. The DNA was then transferred to a nylon filter and hybridized with a 32_{P-labeled fragment containing}

orf167. M, Molecular markerl DNA digested withHindIII andfX 174 DNA digested withHaeIII.

Fig. 2. Physical map of the restriction sites of theCoix1.4 kb BglII/BglII fragment. Genes are represented by dark boxes. The four probes used in Northern and Southern blotting are indicated and correspond tonad3 (probe 1),rps12 (probe 2), tRNASer _{(probe 3) and pseudo-tRNA (probe 4). The arrows}

indicate gene orientation. The restriction sites are: A, A6aI; Bg,BglII; E,EcoRI; Ps,PstI; Pv,P6_{uII; S,SstI; S,SpeI; X,}

XhoI. ThePstI andSstI sites are from the BlueScript plasmid vector, from which 1.4BglII/BglII was cloned.

Fig. 3. Nucleotide sequence of the tRNASer_{/pseudo-tRNA/nad3/rps12 locus in Coix mtDNA (GenBank accession number} AF239263). The amino acid sequence deduced from the nucleotide sequence is shown in single letter code above the nucleotide sequence. RNA editing sites are indicated by a lowercase ‘c’. The predicted amino acid before and after editing are indicated. Horizontal arrows, synthetic oligonucleotides prepared for cDNA synthesis and PCR amplification; dashed line, pseudo-tRNA insertion; stars, stop codons. Pseudo-tRNA and tRNASer _{are underlined. The sequence used for drps12 synthesis was based on}

a consensus sequence between maize and wheat [21].

rps12 gene from Coix mtDNA. The amplification product was cloned and sequenced, and its se-quence added to that of the 1.4 kbBglII fragment (Fig. 3).

The stringency conditions for heterologous hy-bridizations usingorf167 were chosen for detecting low similarity with mtDNA sequences. Sequence comparisons and alignment between the sequences of orf167 and the BglII fragment revealed that

orf167 displayed 45% similarity with theCoix nad3 gene; this similarity was evenly distributed over the gene sequence and responsible for the hybridization signal as checked by hybridization controls with the

nad3 sequence from wheat (data not shown). This value is to be compared with the more significant 82% similarity found when comparing this same Coix nad3 gene with the actual homologous nad3 from M. polymorpha. Although orf167 has been described as a hypothetical 18.6 KD protein in the

nad3 –nad7 intergenic region, with no relationship to the NAD3 protein, the origin of the similarity with nad3 reported here is intriguing. It may be fortuitous or it may reveal an ancient sequence duplication in the mitochondrial genome of M.

polymorpha having occurred in the vicinity of the actual nad3 sequence.

The nucleotide sequences of the Coix nad3/

rps12 gene show 100 – 99.4 and 100 – 99.7% simi-larity with the same gene sequences from maize and wheat, respectively. In Coix, the intergenic spacer sequence between nad3 and rps12 is 44 bp long, very similar to the 47 bp intergenic spacer found in maize and wheat [21].

The Coix tRNASer _{sequence has 100% identity}

with the same gene from maize, wheat and rice [21,24,31]. There is a pseudo-tRNA sequence

lo-cated 259 bp downstream of the trnS gene,

ho-mologous to the sequence found in maize, wheat and rice mitochondrial genomes [21,24,31]. The

Coix pseudo-tRNA gene presents a 50 bp

inser-tional sequence in the same position as in other monocots examined so far (Fig. 3), which is 99% similar to a 48 bp insertion found in the maize pseudo-tRNA. As pointed out previously [24], the rice pseudo-tRNA gene seems to have evolved from tRNAPhe _{because of its high sequence}

ho-mology with tRNAPhe _{[32]. Sequence comparisons}

excluding insertional sequences have shown that

Coixand maize pseudo-tRNA genes are 93%

sim-ilar with the potato tRNAPhe_{. Thus, in all}

mono-cots examined so far [21,24,31], this pseudo-tRNA gene seems to have been inactivated in the course of evolution. This inactivation may have origi-nated from an intramolecular recombination fol-lowed by a sequence insertion in a common ancestor sequence, probably the mitochondrial

tRNAPhe _gene.

Southern blot hybridizations using thenad3 and

pseudo-tRNA genes as probes against Coix,

maize, cauliflower, potato, soybean, pea and al-falfa mitochondrial genomes digested by BamHI, showed that the nad3 gene is present as a single

copy in mtDNA from Coix and other species

(data not shown). However, of the presented

spe-cies, only the maize and Coix mtDNAs gave

strong hybridization signals with pseudo-tRNA gene (data not shown). These results suggest that this pseudo-tRNA gene originated from a recom-bination event that took place in a monocot an-cestor, after the dicot/monocot divergence. 3.3. Transcription of the

trnS/pseudo-tRNA/nad3/rps12 locus

Northern blot hybridizations were carried out

with total mitochondrial RNA from Coix using

probes corresponding to each of the genes in the cluster: a 0.5 kb P6_{uII/SpeI fragment (probe 1)} for nad3, a 0.3 kb SstI/XhoI fragment (probe 2) for rps 12, and a 0.6 P6_{uII/PstI fragment (probe} 3) for trnS (Fig. 2). Only a single abundant tran-script of approximately 1250 nts was detected with probes 1 and 2, indicating that the Coix nad3 and rps12 genes were co-transcribed, as confirmed by RT-PCR amplification (see later). This tran-script pattern was similar to that of nad3/rps12 transcription in rice, where a single 1.2 kb tran-script was identified [24]. However, maize and wheat have shown a more complex transcription pattern, with two and at least four transcripts, respectively [21], and the mRNA is 0.9 kb in length. The mRNA transcript size difference be-tween Coix and maize was surprising, considering the high sequence identity (95 – 98%) shared be-tween the 1455 pb Coix trnS/pseudo-tRNA/nad3/

rps12 locus and the same region in maize and

wheat. Considering that the two mapped 5% _ends of the wheat transcript [21] are within a sequence region showing almost 100% identity with the

Coix sequence, it can be supposed that the differ-ence observed in transcript size would be ex-plained by the 3%_{end transcript change. However,} ends of the nad3/rps12 Coixtranscript need to be mapped to confirm this hypothesis.

Northern hybridizations were used to test

whether the trnS gene and pseudo-tRNA were

transcribed in Coix. In this way, probes 3 and 4 (Fig. 2) were hybridized with Coix total mtRNA.

Fig. 4. RNA hybridizations of thenad3, rps12 and tRNASer

transcripts in Coix. Five micrograms of total mtRNA were denatured and applied to each lane of a 1.2% agarose/ formal-dehyde gel, transferred to a nylon membrane and hybridized

32_{P-labeled fragments ofnad3 (1),rps12 (2) andtRNA}Ser_(3).

An RNA ladder (9.5 – 0.24 kb, BRL) was used to estimate molecular size.

Table 1

Degree of editing of the nad3 andrps12 genes in the 23 cDNA samples analyzed nad3

Editing rps12

CDNA Number of edited sites

CDNA Number of edited sites

clones clones

% N

N %

26 20

Completely edited 6 12 53 6

Incompletely but extensively edited 10 43 18–19 4 17 5

Barely edited 2 9 1 3 13 2

22 0 4

5 17

Completely unedited 0

23

Total 23

Only probe 3 hybridized to a transcript (Fig. 4), indicating that trnSis transcribed inCoixwhereas the pseudo-tRNA is not.

As already mentioned, nad3-rps12 co-transcrip-tion was confirmed by RT-PCR amplificaco-transcrip-tion. Three primers (fnad3, dnad3 and drps12) were designed from the coding regions of the nad3 and

rps12 genes (Fig. 3). An approximately 800 bp amplification product was obtained for the nad3/

rps12 cotranscript.

3.4. RNA editing pattern of the nad3/rps12

transcripts in Coix

To characterize the extent and sites of RNA editing in nad3/rps12 transcripts, cDNA covering the coding region was obtained by RT-PCR using the primers fnad3 or dnad3 and drps12 (Fig. 3). The comparison of the genomic and 23 cDNA revealed 20 C-to-U RNA editing events in nad3 and six in therps12 coding region (Fig. 3). Twenty-six C-to-U editing sites resulted in 16 and Twenty-six codon modifications in thenad3 and rps12 genes, respec-tively, corresponding to 13.6% of the NAD3 and 4.8% of the RPS12 amino-acid sequence. Most of the editing sites in theCoix nad3 –rps12 genes have already been found in wheat [21], Oenothera [33],

Pinus [27], Magnolia, onion, sunflower [34], Petu

-nia [35], Sorghum [23] and Brassica [16]. A com-parison of the nad3 editing positions in Coix and wheat [36] showed four differences: codons 13, 42 and 46 were edited only in wheat, and codon 62 (editing site six in Fig. 3) only inCoix, whereas the

rps12 editing sites in Coix and wheat were identi-cal. Contrary to the case with wheat, these four editing events inCoix nad3 were identical to those

inOenothera[33] and only one of them was differ-ent inPinus[27]. These sites probably existed prior to the evolutionary separation of monocots and dicots, and the species-specific differences origi-nated from genomic point mutations in some lin-eages during evolution.

Sequence analysis of 23 cDNA clones revealed unedited and partially edited clones with no evi-dent polarity for the editing process. The extent of editing differed between nad3 and rps12 tran-scripts: more than 50% of them presented com-pletely edited rps12, whereas only 26% of them were fully edited in thenad3 region (Table 1). This finding suggests that the rps12 editing sites inCoix

are edited first or faster than thenad3 editing sites. This pattern differs from that in Magnolia where all 23 editing sites in the PCR-derivednad3 cDNA were found to be fully edited, while all of the editing sites in rps12 were only partially altered [34]. Mixtures of partially or differentially edited cDNA clone populations derived from the nad3 –

rps12 loci have also been found in the mitochon-dria of other plants such as wheat [36] and pine [27].

In conclusion, a high conservation of the trnS/ pseudo-tRNA/nad3/rps12 sequence, gene organi-zation and editing pattern of thenad3/rps12 genes among Coix and monocots were found, especially between maize and Coix, indicating that evolution has produced a high degree of conservation in this locus in these species. This fact is surprising consid-ering; first, Coix is a distant relative of maize (an Asian species of the Andropogoneae tribe); second, the high recombinationary ability of the mitochon-drial genome plant; and, finally, the general non-conservation of different gene clusters, even in very close evolutionarily species.

Acknowledgements

The authors wish to thank Dr. M.-C. Boisselier-Dubayle (Museum National d’Histoire Naturelle, Laboratoire de Cryptogamie, Paris, France) for

the gift of Marchantia polymorpha total DNA.

This work was financed by grants to A.P.S. from Fundac¸a˜o de Amparo a` Pesquisa de Sa˜o Paulo (FAPESP; 96/03520-8). A.P.S. was also the recipi-ent of a research fellowship from Conselho

Na-cional de Desenvolvimento Cientı´fico e

Tecnolo´gico (CNPq). S.M.G.D. and S.F.S. were supported by graduate fellowships from FAPESP.

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[10] W. Schuster, The highly edited orf206 in Oenothera mitochondriamay encode a component of a heme trans-porter involved in cytochrome c biogenesis, Plant Mol. Biol. 25 (1994) 33 – 42.

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[12] W. Jekabson, W. Schuster, Orf250 encodes a second subunit of an ABC-type transporter inOenothera mito-chondria, Mol. Gen. Genet. 246 (1995) 166 – 173. [13] M. Nakazono, Y. Ito, N. Tsutsumi, A. Hirai, The gene

for a subunit of an ABC-type heme transporter is tran-scribed together with the gene for subunit 6 of NADH dehydrogenase in rice mitochondria, Curr. Genet. 29 (1996) 412 – 416.

[14] H. Handa, G. Bonnard, J.M. Grienenberger, The rape-seed mitochondrial gene encoding a homologue of the bacterial proteinCcl1 is divided into two independently transcribed reading frames, Mol. Gen. Genet. 252 (1996) 292 – 302.

[15] R. Menassa, N. El-Rouby, G.G. Brown, An open read-ing frame for a protein involved in cytochromec biogen-esis split into two parts inBrassicamitochondria, Curr. Genet. 31 (1997) 70 – 79.

[16] K. Itani, H. Handa, Rapeseed mitochondrial ccb206, a gene involved in cytochrome c biogenesis, is co-tran-scribed with the nad3 and rps12 genes: organization, transcription, and RNA editing of the nad3/rps12/

ccb206 locus, Curr. Genet. 34 (1998) 318 – 325.

[17] D. Falconet, B. Lejeune, F. Quetier, M.W. Gray, Evi-dence for homologous recombination between repeated sequences containing 18S and 5S ribosomal RNA genes in wheat mitochondrial DNA, EMBO J. 3 (1984) 297 – 302.

[18] M.D. Hunt, K.J. Newton, The NC3 mutation-genetic-evidence for the expression of ribosomal-protein genes in Zea maysmitochondria, EMBO J. 10 (1991) 1045 – 1052. [19] P.S. Covello, M.W. Gray, Sequence-analysis of wheat mitochondrial transcripts cappedin6_{itro: definitive}

iden-tification of transcription initiation sites, Curr. Genet. 20 (1991) 245 – 251.

[20] M.M. Bland, C.S. Leavings III, D.F. Matzinger, The tobacco mitochondrial ATPase subunit 9 gene is closely linked to an open reading frame for a ribosomal protein, Mol. Gen. Gen. 204 (1986) 4 – 8.

[21] J.M. Gualberto, H. Wintz, J.H. Weil, J.M. Grienen-berger, The genes coding for subunit 3 of NADH dehy-drogenase and for ribosomal protein S12 are present in the wheat and maize mitochondria genomes and are co-transcribed, Mol. Gen. Genet. 215 (1988) 118 – 127. [22] A.W. Liu, K.K. Narayanan, C.P. Andre´, E.K. Kaleikau,

V. Walbot, Co-transcription oforf25 andcoxIIIin rice mitochondria, Curr. Genet. 21 (1992) 507 – 513. [23] W. Howad, F. Kempken, Sequence analysis and

tran-script processing of the mitochondrialnad3 –rps12 genes from Sorghum bicolor, Plant Sci. 129 (1997) 65 – 68. [24] T. Suzuki, S. Kazama, A. Hirai, T. Akihama, K.

Kad-owaki, The rice mitochondrial nad3 gene has an ex-tended reading frame at its 5%_{end: nucleotide sequence}

analysis of ricetrnS,nad3, andrps12 genes, Curr. Genet. 20 (1991) 331 – 337.

[25] P. Brandt, S. Su¨nkel, M. Unseld, A. Brennicke, V. Knoop, The nad4L gene is encoded between exon c of nad5 and orf25 in the Arabdopsis mitochondrial genome, Mol. Gen. Genet. 236 (1992) 33 – 38.

[26] C.A. Makaroff, J.D. Palmer, Mitochondrial DNA rear-rangements and transcriptional alterations in the male sterile cytoplasm of Ogura radish, Mol. Cell Biol. 8 (1988) 1474 – 1480.

[27] B. Karpinska, S. Karpinski, J.E. Hallgren, The genes encoding subunit 3 of NADH deydrogenase and riboso-mal protein S12 are co-transcribed and edited inPinus syl6estris (L.) mitochondria, Curr. Genet. 28 (1995) 423 – 428.

[28] F. Vedel, F. Quetier, Physico-chemical characterization of mitochondrial DNA from potato tubers, Biochim. Biophys. Acta 340 (1974) 374 – 387.

[29] D.B. Stern, K.J. Newton, Isolation of plant mitochon-drial RNA, Methods Enzymol. 118 (1986) 488 – 496. [30] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular

cloning: a laboratory manual, 2nd ed. (1989), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [31] H. Wintz, J.M. Grienenberger, J.H. Weil, D.M.

Lons-dale, Location and nucleotide sequence of two tRNA genes and a tRNA pseudogene in maize mitochondrial genome: evidence for the transcription of a chloroplast gene in mitochondria, Curr. Genet. 13 (1988) 247 – 254. [32] L. Mare´chal, P. Guillemault, J.M. Grienenberger, G. Jeannin, J.H. Weil, Structure of bean mitochondrial

tRNAPhe _{and localisation of the tRNA}Phe _{on the}

mito-chondrial genomes of maize and wheat, FEBS Lett. 184 (1985) 289 – 293.

[33] W. Schuster, B. Wissinger, M. Unseld, A. Brennicke, Transcripts of the NADH-dehydrogenase subunit 3 gene are differentially edited in Oenothera mitochondria, EMBO J. 9 (1990) 263 – 269.

[34] G. Perrotta, T.M. Regina, L.R. Ceci, C. Quagliariello, Conservation of the organization of the mitochondrial nad3 and rps12 genes in evolutionary distant an-giosperms, Mol. Gen. Genet. 251 (1996) 326 – 337. [35] B. Lu, M.R. Hanson, A single nuclear gene specifies the

abundance and extent of RNA editing of a plant mito-chondrial transcript, Nucleic Acids Res. 20 (1992) 5699 – 5703.

[36] J.M. Gualberto, G. Bonnard, L. Lamattina, J.M. Grienenberger, Expression of the wheat mitochondrial nad3 –rps12 transcription unit: correlation between edit-ing and mRNA maturation, Plant Cell 3 (1991) 1109 – 1120.