The highly edited orf206 in Oenothera mitochondria may encode a component of a heme transporter involved in cytochrome c biogenesis

Plant Molecular Biology 25: 33-42, 1994. © 1994 Kluwer Academic Publishers. Printed in Belgium. 33

The highly edited orf206 in Oenothera mitochondria may encode a component of a heme transporter involved in cytochrome c biogenesis

Wolfgang Schuster Institut fiir Genbiologische Forschung, Ihnestrasse 63, D-14195 Berlin, Germany

Received 28 September 1993; accepted in revised form 7 February 1994

Key words: RNA editing, plant mitochondria, cytochrome c assembly, Oenothera

Abstract

A highly transcribed region in Oenothera mitochondria codes for a reading frame (orf206) which shows high homology to the Marchantia encoded mitochondrial open reading frame orf277 and is also conserved in the mitochondrial genomes of Arabidopsis thaliana and Daucus carota. Transcripts of orf206 are modified by cytidine to uridine changes in 46 positions by RNA editing, affecting 30~o of all cytidines and 15 ~o of the total encoded amino acids. This ORF is cotranscribed with an upstream reading frame and with the downstream rpsl4 gene. The orf206 deduced protein shows high similarity to polypeptides which are proposed to be part of an ABC-type heine transporter involved in cytochrome c biogenesis in Bradyrhizobium and Rhodobacter.

Introduction

The genes encoded in mitochondrial genomes re- present the residual genetic information of the original endosymbiontic bacterium [ 7 ]. This mitochondrial genetic information varies considerably between different organisms. While eight reading frames are present on the 15 kb Chlamydomonas mtDNA [14] and 13 protein coding genes are found in the 16 kb animal mitochondrial genomes [4], the 187 kb mtDNA of the liverwort March- antia contains 62 putative open reading frames (ORFs) [17]. We have approached the question of the actual gene content of the still larger mitochondrial genomes of higher plants with sizes

between 200 and 2400kb [26] by specifically investigating transcribed regions on the mitochondrial genome of higher plants with respect to potentially relevant coding regions. This approach circumvents most of the plastid and nuclear sequence insertions in the plant mitochondrial genome, which contribute in part to the expan- sion of these genomes, but are rarely transcribed.

One of the transcribed mitochondrial regions thus identified was found to encode a protein involved in cytochrome c biogenesis [22], related genes of which are found in the photosynthetic bacterium Rhodobacter [3] and in the symbiotic Gram-negative bacterium Bradyrhizobium [ 19]. These prokaryotes belong to the bacterial group

The nucleotide sequence data reported wile appear in the EMBL, Genbank and DDBJ Nucleotide Sequence Databases under the accession number X74164

34

considered to be phylogenetically related to the ancestral mitochondrial endosymbiont [8]. In Rhodobacter eapsulatus six genes at two loci have been described that are required for cytochrome c biogenesis in prokaryotes [3 ]. One of these loci encodes two periplasmically oriented proteins (ccll and ccl2), the second locus contains five genes (helA, helB, heIC, helD and helX), which were proposed to encode an ABC-type heme transporter (helA-helD), and a disulphide oxi- doreductase (helX) required to mediate distinct oxidation or reduction reactions [2].

The open reading frames (ORFs) previously identified in Oenothera (orf577), carrot (orf579), and wheat (orf589) mitochondria [6] are at least partial homologues of ccll. In Marchantia polymorpha this open reading frame is part of a mitochondrial gene cluster of which at least five genes show high similarity to three of the Rhodo- bacter and Bradyhizobium genes hel, cyc and ccl [3, 19]. This clustered organization does not appear to be conserved in mitochondria of the higher plant Oenothera, where the ccll homologue is not connected with other genes of this cluster. The results described here identify a second gene of this bacterial cistron in Oenothera mitochondria to be located upstream of the ribosomal protein gene rpsl4 [25]. The identification of this second gene in higher plant mitochondria with high similarity to proteins involved in bacterial cytochrome c biosynthesis suggests that in plants possibly even more genes of this bacterial cluster have not yet been translocated to the nuclear compartment, but that the individual genes retained have been scattered around the mitochondrial genome by the frequent recombination events in higher plant mitochondria.

Materials and methods

Isolation of mitochondria

Mitochondria of Oenothera berteriana, Daucus carom and Arabidopsis thaliana were isolated from tissue and suspension cultures (A. thaliana) by differential centrifugation followed by Percoll gra- dient centrifugation as described previously [24].

Cloning of mtDNA and cDNA

Purified mitochondria were digested with DNaseI and after several washing steps were lysed in 0.5 ~o SDS followed by CsC1/ethidium bromide gradi- ent centrifugation. This highly purified mtDNA was cloned into pBR322 and pUC vectors by standard techniques [20]. Construction of the Oenothera mitochondrial cDNA library has been described in detail [27].

Sequence analys~

Fragments of interest were subcloned into pUC and Bluescript (Stratagene) vectors for construction of partially deleted subclones. Deletion cloning was performed by the Exonuclease III method using the nested deletion kit supplied by Pharma- cia. Sequences were determined by chain termi- nation [21] in the dideoxy double-stranded sequencing procedure (Pharmacia) with T7 polymerase. PCR derived cDNA was directly se- quenced. Both strands of the genomic sequences were analysed and sequences were compared and aligned with the program FASTA [ 13, 18].

Southern and northern hybridization

Total mtDNAs of Oenothera berteriana, Daucus carota and Arabidopsis thaliana were digested with restriction enzymes, fractionated by electrophore- sis in agarose gels and transferred to Biodyne A membranes (Pall). DNA hybridization was carried out in standard buffers [20], and washes were performed at 60 °C in 0.1 x SSC and 0.1Yo SDS. Filters were autoradiographed at -80 °C using X-ray film. Total mtRNA was fractionated in 1.5~o formaldehyde/agarose gels and transferred to nylon membranes using standard pro- cedures [20]. Hybridization and washes were carried out under standard conditions [20].

PCR amplification

To obtain specific cDNA sequences, random hexamer-primed reverse transcribed mtRNA of Oenothera was used as template for the PCR

35

H

I B X H H H B

I 'oo I I cDNA clones

, / / "

. / " / ..

• ~..'.'.'..:.'.'..:." :.'." :.k

Fig. 1. Genomic organization of the orf206 reading frame in Oenothera mitochondria. The upper part shows the two loci encod- ing the orf206 gene. The sequences diverge downstream of the inverted repeat region 3' of which in one allele the rps14 and cob genes are located. Position and extent of independent cDNA clones covering the orf206 and surrounding regions are shown by horizontal lines. PCR amplification of the orf206 cDNA is indicated by arrowheads and dotted lines below the cDNA clones. RNA editing sites identified in orf206 transcripts are given as vertical arrows above the enlarged gene region. Restriction recognition sites are indicated for Barn HI (B), Hind III (H), and Bst XI (X).

reactions. PCR amplifications were done with combinations of the following primers: 5' primer, 5' -ATAGGATCCATGAGAAGACTCTTTCT- TG-3 ' ; 3' primer, 5 ' - A G G A G A A T T C G C C A T - TCAATCTTG-3 '

The reaction mixture contained 50 mM KC1, 1.5 mM MgC12, 10 mM Tris-HC1 pH 8.3, 500 ng of each primer, 50 fmol of each dNTP, 10 ng cDNA and 5 units of Taq polymerase (Boe- hringer). PCR was performed on a Biomed cycler under following conditions: cycle 1, 1 min at 94 °C; cycle 2, 1 min at 45 °C, cycle 3, 3 rain at 72 °C. All cycles were repeated 30 times and an extension of 10 min at 72 °C was added at the end. After the PCR reaction an aliquot of the product was analysed on an agarose gel. The Bam HI/Eco RI-digested PCR product was further purified and cloned into a Bluescript (Stratagene) vector.

Results

Cloning, genomic location, and sequence of orf206

A highly transcribed region in the Oenothera mitochondrial genome, upstream of rps14, has been

Fig. 2. The two alleles of the orf206 gene are identified in Oenothera mitochondrial DNA. Southern hybridization of BamHI and Hind III digests of mtDNA of Oenothera with the Bam Hl/Bst XI fragment covering most of the orf206 reading frame shows two fragments each with 1.4 and 4.2 kb for Barn HI, and 1.5 and 3.0 kb for Hind III. Sizes of length markers are shown on the left in kb.

36

identif ied by hybr id iza t ion wi th label led reverse

transcr ibed m t R N A . Over lapping c l o n e s cover - ing this region were i so la ted f rom Oenothera m t D N A libraries by hybr id iza t ion wi th the Bam HI/Bst X I fragment cover ing the 3' part o f orf206. A s e q u e n c e o f 106 nuc l eo t ides l o c a te d b e t w e e n rpsl4 and this O R F is c o n s e r v e d a lso at

the 3' ends o f atpA and coxH transcripts in Oenothera m i t o c h o n d r i a . This s e q u e n c e can form an inverted repeat and is m o s t l ikely invo lved in m R N A stabil ity o f m a n y m i t o c h o n d r i a l genes [23 ]. D o w n s t r e a m o f this s e q u e n c e a r e c o m b i n a - t ion event has c o n n e c t e d o n e c o p y o f orf206 with a different e n v i r o n m e n t (Fig. 1). Southern blot

ATGTTACGCGGCGTTCAG~C~AGCCTTGAAGTTTTTGAATTTGAAAG~GT~AAAA 58 orf206~ M R R L F L E L Y [ - H - - ~ K Q I I I F I P ~ S I

i I t i

TccAcAccA ACO O TTTTC 'CC CI COITA 'CIO CI14 v • p L M L G v E K D F IS~L 1 C H

GTA ACG CCC TTA ATG CTA GGT TTT G~ AAA GAC TTTBTCABTGT CAT 193

C T TTA GGT ATT TGG ATC CTG TTG TTT CCT TTT 238

P F IP~Fi R N E K E D G T L E

~ C_CC~GCA CCT TTT CGA AAT GAG AAA GAA GAT GGT ACA CTC GAA 283

L Y Y L S A Y C L P K I L L L TTG TAT TAT TTA AGT GCT TAT TGC TTG CCA A~ ATC CTA CTT CTA 328

Q L V G H W V I Q I S ~ V F C CAA TTG GTA GGT CAC TGG GTT ATT CAA ATA AGT ~ GTT TTC TGT 373

o s

/

C G I H S R~CIS~L 1A L G I T S S ICTGBTGT GGT ATT CAT TCT CGTITCGIGCT CTT GGA ATA ACA TCA AGC 508

1 1

AGT GGT TGG AAC AGC TCG I 553

L P P~L T L S~F R~C I T S I E T E W F TTG CCC CCA ACT CTT TCT CGT ACC TCT ATT GAA ACA GAA TGG TTT 598

i

p ~ is~Ll v s ~ s Is~L[ ~ n * TTT CCA ATTiTCGIGTC TCG ATT AGTITCABCAA GAT TGA ATGGCCAATTCT 691

CCTC~GGACCCTCGATTCGATTGTTTTTCAAGAATGTCCGGGTATGTAAGCCATGTAT 749

CTGGGAGGAACAACAAGGGTAGTCTTTTTCTCACGAAGAAGATGAGGCCCGCCCGAAAGA 809

AGAAGAAGCGGGCGGGGTGAAAGAGTGGGGATGAGGGCTTTCACGTGTGCTATAATAGGT 869

GGGAGATGATCTCCGATCCCGTAACAGAGTGGTGCTTTAGCGAGTCGCTTTCCGCTCCGG 929

ctttgttgttagttctcctgactcgatactttgagaactacagt

GCTCCATACGTTTACT ACGACCGTGG~TACTTTTAATTTAGAAAAG~GAACT CAA 987

rpsl4~ M E K R N I R D H K R R L L A

ATG GAA AAG CGA AAT ATA CGA GAT CAC AAA CGT AGA TTG CTC GCG 1032

Fig. 3. RNA editing sites in the o~06 gene. The sequence shown covers the ORF, the transcribed spacer, and the sequence divergence of the two alleles. The locus containing the 5' pa~ of the ~s14 gene is continued in capital letters, while the sequence of the second allele is ~ven in lower-case letters downstream of the point of divergence. Translation of the open reading ~ame is shown above the sequence and the inverted repeat moti~ conserved downstream of several other genes, is underlined. Cytidines altered by RNA editing are underlined and triplets are boxed with the respective concurrent amino acid alterations indicated.

37

hybridization with the Bam HI/Bst XI fragment covering a large part of the orf206 reading frame identifies two Bam HI fragments of 4.2 and 1.4 kb and two Hind III fragments of 1.5 and 3.0 kb, respectively (Fig. 2). Both hybridizing fragments have been isolated from mtDNA libraries and sequence analysis of the two genomic loci revealed the point of divergence to be located 269 nucleotides downstream of orf206 (Fig. 3). The sequence downstream of this point of divergence revealed no homology to sequences in the databases.

Transcription of orf206

Analysis of cDNA clones indicates that orf206 is cotranscribed with the rpsl4 gene, which begins 311 nucleotides downstream. Northern blot hybridization experiments with total mitochondrial RNA from Oenothera probed with an internal

orf206 probe revealed a complex transcription pattern confirming the cotranscription of o~7~206 with an additional upstream ORF and the downstream located rpsl4 gene (Fig. 4); with transcripts ranging from 1.0 kb to 6.5 kb in size. cDNA clones spanning this genomic region have been isolated. The most prominent transcript is 2.3 kb and covers orf206 and the upstream region, which contains an ORF that shows high similarity to part of rpl2 (not shown). This rpl2 reading frame is interrupted by a group II intron, that is spliced in all cDNA clones covering orf206 and this upstream reading frame (unpublished results). The inverted repeat between orf206 and rpsl4 is present in several cDNA clones suggesting that this structure, which is also found at the 3' mRNA terminus ofatpA and coxH transcripts in Oenothera [23, 25], is not an effective termina- tor of transcription but may rather be involved in stabilizing 3' ends of RNAs. It is still unclear whether the second locus of orf206 without the downstream rpsl4 gene is transcribed, because no cDNA clone with sequences 3' of the inverted repeat has been found.

Editing sites of Oenothera orf206 transcripts

Fig. 4. Northern hybridization of Oenothera mtRNA with the Bam HI/Bst XI fragment. A complex transcription pattern of more than 8 transcripts is observed with RNA molecule species of between 1.0 and 6.5 kb in length. RNA molecular weight markers are given on the left in kb.

RNA editing in mitochondria of higher plants [ 5, 9, 10] modifies the sequence on the RNA level by C-to-U conversions. To identify the extent of RNA editing in transcripts of orf206, cDNA clones covering the coding region were isolated from a cDNA library and complemented by PCR analysis of total mitochondrial RNA. Direct sequencing of PCR derived cDNA gave no indica- tion of partially edited sites. Differences between the genomically encoded and cDNA-derived sequences show 46 cytidines in the genomic sequence to be altered to uridines in the RNA sequence of orf206 (Fig. 3). These C-to-U modifi- cations result in 31 amino acid changes (15 ~o ) of the total of 206 encoded residues, all of which improve the similarity to homologous polypeptides of other organisms (Fig. 4). Fifteen serine, eleven proline, four arginine, and one histidine triplets are changed by editing, resulting in a more

38

Oenothera gen. H P

Oenothera eDNA R ~ H ~ S - ~ Marchantia mt MKRVRE ENET LHLENARRSPPLAS THFLGFPC I SLFYSQHKSTKKN IYLD LKTKKKE LLPMVFAL LN Rhodobacter helB ~A~-~SRDLRLAIR--AG Bradyrhizobium cycW ~A~- IRRDIRIA~R--VG

P S S S P P pp p R

r.=.~m,*.- -~i~:,.=~.=..,.n,~4Nms - ~ . . . . . . umJ~zl~mwmJaa GGFGLG~AFF vL~LV~FGV~PQGE I~AR I AS~I L~LGA~LAC 14~LD~I~AL~S~D~LATAP I PMEAVVT I~AL~I T TG LP LVLAA~LFAV~LH L GGA L I GV LFF LT~V LM~FAV~PD LAL~S RLGPA I L~LGA~LAS L~T L D ~ ~ L A ~ L A A G LP LVATPV LG -[R]JLN L

R P RS S S S S P SR PSS SSP S S

S L~T P A~S V~G T FGAA~TV~LKRGG L LL~L L V~P L YV~I FGAE~VRRGA~G LA I E~P LAM~A~I T AA ~ AS A~NLR * ~MVATGAVALTL~TPA~FTGMHGAA~VTLHRGGLLMAVLV~PLS I~I FGVAASQAVA IVGPMS FGAPFS I LCA~S~IGPFAAAA~RHG~D*

Fig. 5. The Oenothera mitochondrial orf206 is homologous to the Marchantia o0C277 and to the bacterial genes helB and cycW. Translations of the genomic (gen.) and eDNA sequences from Oenothera or/206 are aligned with the respective polypeptides from Marchantia (orf277) [17], Rhodobaeter (helB) [3], and Bradyrhizobium (cycW) [19]. For the Oenothera genomic DNA derived polypeptide only amino acids altered by RNA editing are indicated. Gaps introduced for improved alignment are shown by dashes. Amino acids conserved to the edited Oenothera orf206 deduced sequence are highlighted by inverse contrast.

hydrophobic protein. 00c206 transcripts are the most highly edited mRNAs observed to date in plant mitochondria [28]. About 30~o of all cytidines are edited in this reading frame, including nine silent edits without influence on the amino acid sequence. Two additional editing sites were identified 37 nucleotides upstream of the translation start site and 17 nucleotides downstream of the stop codon of the orf206 reading frame.

The deduced ORF206 polypeptide is homologous to bacterial genes required for cytochrome c assembly

As is shown in Fig. 5, the deduced amino acid sequence of orf206 shows significant similarity to polypeptides conserved in the lower plant March- antia polymorpha [17] and in the bacteria Bradyrhizobium and Rhodobacter [3, 19]. Com- parison of the edited m R N A deduced protein of orf206 shows 76~o identical amino acids with Marchantia orf277. The Marchantia open reading frame (ORF) is extended at the amino terminus by 66 amino acids, which are present neither in the higher plant mitochondrial nor in the bacterial polypeptides. In the Oenothera orf206, 26 ~o of the amino acids are identical with the Rhodobacter helB and 21 ~o are identical with the Bradyrhizo- bium cycW-encoded proteins.

Fig. 6. Orf206 is conserved in the mitochondrial genomes of higher plants. Hybridization with the Bam HI/Bst XI fragment reveals the presence of orf206 on single restriction fragments in Bam HI digests of the mitochondrial DNAs of Ara- bidopsis thaliana and Daucus carota and the two alleles in the Oenothera mtDNA. Sizes of length standard fragments are given in kb on the left.

cf206 Oenothe:

"_f277 Marchant:

-2-

-3-

0 50 100 150 200

100 150 200 250

0 50 100 150 200

2

1

0

-i

"2

3IB Rhodobact~ 3

50 100 150 200 3

m :o+ ~m ~o

3

2

1

0

-1

3

39

Orf206 appears to be a general structural gene of plant mitochondrial genomes, since it is also conserved in other plant species. Hybridization with an orf206-specific probe to mtDNAs of other higher plants (Fig. 6) revealed that in Arabidopsis this gene is localized on a unique 10 kb Bam HI fragment, and likewise in Daucus carota there is a single copy of this gene on a 2.2 kb Barn HI fragment.

The orf206 gene codes for a hydrophobic protein (Fig. 7) and shows a strikingly similar hydrophobicity pattern to the analogous polypeptides of Marchantia, Rhodobacter and Bradyrhizobium. Such highly periodic hydrophobic profiles are typical of integral membrane proteins with transmembrane helices.

Discussion

In this report a new mitochondrial gene (orf206) in higher plant mitochondria is identified which is homologous to the mitochondrially encoded orf277 in Marchantia polymorpha [17]. In Oenothera mitochondria, this open reading frame is part of a transcription unit which encodes upstream of orf206 the N-terminal part of the ribosomal protein L2 and downstream (in one ar- rangement) the ribosomal protein S 14. This gene order is not conserved in the Marchantia mitochondrial genome, where orf277 (the orf206 counterpart) is part of a cistron that encodes several open reading frames with high similarity to the bacterial gene clusters hel, ccl and cyc [3, 19].

Another gene (orf509) of this mitochondrial cistron of Marchantia is conserved in the higher- plant mitochondrial genomes of Oenothera (orf577), carrot (orf579) [22] and wheat (orf589)

Fig. 7. Hydrophobicity analyses of orf206, orf277, helB and cycW. Hydrophobicity analyses were performed using a win- dow length of 19 residues [ 12] with the G C G programs. Re- gions < 0 are hydrophilic, regions > 0 are hydrophobic, with regions more than 1.6 being predicted as transmembrane regions [12]. The distribution of hydrophobic regions in the respective proteins of plant mitochondria and bacteria is very similar confirming homologous functions for these polypeptides.

40

[6]. This gene shows high similarity to the N-terminal part of the Rhodobacter gene ccll, the C-terminal part of which is encoded by two additional open reading frames (orf169 and orf322) in Marchantia, which are also conserved in higher plant mitochondrial genomes (unpublished results). Altogether now five ORFs in the mitochondrial genome of Marchantia polymorpha, or- ganized in one cluster, have been identified which show significant homologies to bacterial genes that are important for the biogenesis of c-type cytochromes.

Cytochromes of the c-type consist of an apo- protein to which a heme cofactor is covalently bound. These cytochromes function as redox proteins in the mitochondrial electron transfer chain as well as in the respiratory chains of many bacteria. Mitochondria contain two c-type cytochromes, a soluble cytochrome c in the intermembrane space and a membrane bound cytochrome Cl oriented to the intermembrane space. Studies performed with mitochondrial cytochrome c have shown that the formation of the thioether linkage does not occur spontaneously under physiologi- cal conditions but is enzymatically catalysed by cytochrome c heme lyase [15]. In eukaryotes it has been shown that at least three components are required in the intermembrane space for assembly of c-type cytochromes: apocytochrome c, heme, and heme lyase [ 16]. In rhodobacter cap- sulatus and Bradyrhizobium japonicum several genes involved in cytochrome c biogenesis have been analysed, among which periplasmic protein disulfide oxidoreductases and components of ATP-binding cassette (ABC)-type heme transporters have been identified [2].

ABC-type transport systems seem to be rela- tively specific for single substrates, which can be small molecules such as amino acids or sugars, but also larger molecules such as polysaccharides or proteins. This superfamily of transporters in- cludes periplasmic binding-protein-dependent uptake systems of prokaryotes, bacterial export- ers, and eukaryotic proteins including the P- glycoprotein associated with multidrug resistance in tumours, and the product of the cystic fibrosis gene [11]. All these permeases have a similar

composition, requiring three membrane-bound components and in the case of the importers one or more periplasmic factors [ 1 ]. One of the membrane components provides the energy require- ment for substrate accumulation by hydrolysis of ATP. This protein, which contains the ATP- binding cassette, is in the case of the heme transporter encoded by the helA gene in Rhodobacter [3] and by the cycV gene in Bradyhizobium [19]. These genes, however, have no counterpart in the mitochondrial genome of Marchantia, and are most likely encoded by a nuclear gene in this m o s s .

The other two membrane-bound proteins of the heme transporter are mitochondrially encoded in Marchantia by orf277 and orf228 [17]. Both reading frames are also conserved in higher-plant mitochondria (data not shown). An N-terminal extension of about 60 amino acids in the moss is not present in the bacterial proteins HelB of Rhodobacter and CycW of Bradyrhizobium and its possible function in Marchantia is still unclear. The second membrane-bound protein of the proposed heme transporter, the helC gene product of Rhodobacter, is also encoded by the mitochondrial genome of the higher plant Oenothera (in preparation).

The reading frames involved in cytochrome c biogenesis are the most highly edited coding regions found to date in higher plant mitochondria. 0rf577 mRNAs of Oenothera are edited in 46 positions, changing 37 amino acids [22], and in wheat mitochondria the corresponding reading frame orf587 is edited in 34 positions [6]. In the here described orf206 RNA editing changes 46 cytidines to uridines, altering 15~o of the 206 amino acids. This reading frame is thus the most highly edited ORF described to date in higher plant mitochondria. Without RNA editing this polypeptide could presumably not be functional, because most of the amino acid replacements are at highly conserved positions of the protein.

In Marchantia the ORFs involved in the cytochrome c biogenesis are clustered [17] with the orientation conserved to the hel and ccl clusters of Rhodobacter. However, this gene organization is not preserved in Oenothera, where these ORFs

have been scattered around the genome to individual locations and transcriptional contexts by the frequent recombination and rearrangements common to higher plant mitochondria. Co- ordinated regulation on the transcriptional level is unlikely to have been maintained for these dis- persed genes, suggesting that regulation has most likely been transferred to the post-transcriptional level for the functionally linked protein products.

Acknowledgements

I thank Dr Axel Brennicke and Dr Charles Andr6 for comments on the manuscript. I also thank Waltraut Jekabsons and Iris Gruska for excellent technical assistance and Dr Volker Knoop for the northern blot. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Bundesministerium f~r Forschung und Tech- nologic.

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Documents

The highly edited orf206 in Oenothera mitochondria may encode a component of a heme transporter involved in cytochrome c biogenesis