Mol Gen Genet (1987) 210:44-51 MG'G © Springer-Verlag 1987

Plastid DNA in the mitochondrial genome of Oenothera: Intra- and interorganellar rearrangements involving part of the plastid ribosomal cistron Wolfgang Schuster and Axel Brennicke Lehrstuhl fiir Spezielle Botanik der Universitfit Tiibingen, Auf der Morgenstelle 1, D-7400 T/ibingen, Federal Republic of Germany

Summary. Part of the plastid rRNA cistron is present in the mitochondrial genome of Oenothera. This sequence of 2081 nucleotides contains the 3' half of the plastid 23 S rRNA, the adjacent intergenic region and the 4.5 S rRNA. Secondary intramitochondrial sequence rearrangements in- volve this region of plastid origin and the gene encoding the putative mitochondrial small ribosomal protein $13. Sequence comparison suggests that the interorganellar transfer event occurred a long time ago. The mitochondrial sequence contains regions more homologous to the plastid DNA from tobacco than from Oenothera itself in the re- gions analysed, suggesting faster sequence evolution in plas- tids than in mitochondria of Oenothera.

Key words: Interorganellar sequence transfer - Plastid 23 S rRNA - Intramitochondrial sequence rearrangement- Oen- o thera - Plant mitochondria

Introduction

A number of examples of transfer of genetic information between different cellular genomes have been described. Many of these involve transfer of sequences from mitochon- dria and plastids to the nuclear genome (for reviews see Timmis and Scott 1983; Gellissen 1987; Gellissen and Mi- chaelis 1987). Events of this nature have been interpreted as supporting the endosymbiont theory of the evolution of plastids and mitochondria which postulates the sequen- tial transfer of genetic information from the prokaryotic ur-endosymbiont to the nucleus of the host cell. Protein transport mechanisms appear to influence the preference for expression of mainly hydrophobic polypeptides in many organellar genomes (Grivell 1983).

The transfer of genetic material however does not seem to operate in a specific direction but rather randomly. The nuclear genomes of those species examined to date contain mitochondrial sequences (and plastid information in higher plants). "Foreign" sequences have also been integrated and maintained in the mitochondrial genomes of those higher plants investigated so far. In the main these are sequences of plastid origin (Stern and Lonsdale 1982; Stern and Palmer 1984; Lonsdale 1985, 1987). Recently however we have shown that nuclear sequences are present in the mito- chondrial genome of Oenothera (Schuster and Brennicke 1987).

Offprint requests to : A. Brennicke

Analysis of these nuclear related sequences reveals an open reading frame with high homology to transposon and viral reverse transcriptase. This observation raises the intri- guing possibility of nucleotide sequence transfer via RNA molecules, that are transported into the foreign organelle and locally reverse transcribed into DNA, which is subse- quently integrated into the organellar genome (Schuster and Brennicke 1987). The presence of this reading frame might be connected with the presence of foreign sequences.

No foreign sequences have been found in highly com- pact genomes such as the mitochondrial DNAs of mammals and insects (Bibb et al. 1981; Clary and Wolstenholme 1985) and chloroplast DNAs in higher plants (Shinozaki et al. 1986). Other genomes with little apparent pressure on compactness such as most nuclear DNAs and the mito- chondrial genomes of higher plants however maintain some of the sequences that have entered their domains and add them to their gene pool.

The selection pressures on some organellar genomes for compactness are as yet unclear. A high mutation rate is characteristic of the compact mammalian mitochondrial ge- nomes, while a slower rate of nucleotide evolution seems to prevail in plant mitochondrial genomes. To compare the rates of sequence evolution in the two plant organelle ge- nomes we determined the divergence of a region common to plastids and mitochondria.

In this report we describe a region which has been trans- ferred from the plastid genome and subsequently integrated in the mitochondrial genome of Oenothera and which con- tains the 3' half of the plastid 23 S rRNA, the adjacent intergenic region and the entire 4.5 S rRNA sequence. We also show intramitochondrial recombination involving the mitochondrial gene for the putative ribosomal protein $13 and the integrated plastid 23 S rRNA sequence. Sequence analysis of these regions and the presumed homologous plastid progenitor suggests faster nucleotide sequence evo- lution in plastids than in mitochondria of Oenothera.

Materials and methods

Mitochondrial nucleic acids were isolated as described (Brennicke 1980; Schuster and Brennicke 1985). Restriction enzyme digests were done as described by the manufacturer (Boehringer Mannheim) and gel techniques followed stan- dard procedures (Maniatis et al. 1982). Sequence analysis was done by controlled chemical modification (Maxam and Gilbert 1980). Computer searches were done by Drs. P.G.

45

P

I I cox I B H

I I I - i -su 9

l k b

B P I I

I C0Xl I

1'

P I A

PB H S13

~' ND 1 Ex,-1 ND 1 Ex-2 !

I PB H

t N'm a-1 .61E -Z 1 i H

B pf/,,S S rRNIA

Fig. 1. Schematic diagram of the Oenothera mitochondrial linkage groups involved in intramitochondrial reorganisation of the gene encoding ribosomal protein $13 (linkage group I) and the integrated sequence from the plastid ribosomal cistron. This major plastid sequence in the mitochondrion contains the 3'-terminal region of the 23 S rRNA, continues through the intergenic spacer and the 4.5 S rRNA and terminates just before the plastid 5 S rRNA sequence (linkage group III). The mosaic linkage group resulting from intramitochondrial rearrangement is shown in linkage group II with the derivation of sequences indicated by homologous hatching. Arrows signify the borders A and B of this rearrangement; the sequence between A and B is identical in linkage groups II and II1 (data not shown). Coding sequences are shown with the orientation 5' to 3' (left to right) above the strand and in opposite orientation below. Gene designations: COX I and COX II, cytochrome oxidase subunits I (Hiesel et al. 1987) and II (Hiesel and Brennicke 1983); SU 9, ATPase subunit 9 (Schuster and Brennicke, in preparation); ND 1 Ex-t and ND 1 Ex-2, NADH dehydrogenase subunit 1 exons t and 2 (Schuster and Brennicke, in preparation); S13, putative ribosomal protein S13 (Fig. 5). Restriction enzyme recognition sites: B, BamHI; H, HindIII; P, PstI

Isaac and C.J. Leaver, Edinburgh. The Oenothera berte- riana chloroplast D N A clones from green leaves were a kind gift of J. vom Stein and W. Hachtel, Bonn. Sequence editing was done with the programs of Lang and Burger (1986).

R e s u l t s and d i scuss ion

As part of our continuing description of the molecular structure of the mitochondrial genome in O. berteriana as a model system for a dicotyledonous higher plant we have analysed and sequenced a number of protein coding genes. We have identified mitochondrial genes by using heterolo- gous probes from maize mitochondria, selected c D N A clones and c D N A of mitochondrially enriched cellular RNA. Examination of one specific locus identified with the latter probe revealed the presence of a plastid sequence in the mitochondrial genome.

Localisation of the plastid sequence

This sequence homology is located upstream of the ATPase subunit 9 gene in the mitochondrial genome of Oenothera (Schuster and Brennicke, in preparation) as indicated in the schematic representation of linkage group III in Fig. 1. A region of 2081 nucleotides (Fig. 2) displays a high degree of homology with plastid sequences encoding the 23 S r R N A and the adjacent 4.5 S rRNA, suggesting the plastid origin o f this region of the mitochondrial genome (Fig. 3). Average homology across the entire fragment amounts to 94% between the mitochondrial D N A of Oenothera and the plastid sequence of tobacco (Shinozaki et al. 1986). To allow an estimate of relative sequence evolution rates be- tween the different organelle genomes we sequenced the

border sections of the Oenothera sequence common to plas- tid and mitochondrial D N A and compared them with each other and with the tobacco plastid sequences.

Comparison of plastid sequences

In order to understand the relationship between the mito- chondrial sequence and the progenitor we analysed the Oen- othera plastid sequences in cloned D N A from chloroplasts isolated from green leaves and from proplastids obtained from pale tissue culture cells that had been maintained in the dark for 10 years. No nucleotide difference between the two plastid types was detected in these sequences (data not shown).

The 23 S, 4.5 S and 5 S r R N A intragenic regions are highly conserved in tobacco and Oenothera plastids as ex- pected for r R N A genes (Fig. 3 and additional data not shown). The spacer regions between the three genes show multiple nucleotide exchanges and sequence duplications/ deletions in the two species. The largest such duplication event has inserted 32 extra nucleotides in the tobacco plastid which have subsequently diverged to some degree.

Comparison of the mitochondrial copy and the plastid sequences

Single nucleotide differences between the tobacco and the Oenothera sequences could have occurred either in the to- bacco plastid or in the Oenothera plastid before transfer of this sequence to the mitochondrion. The divergences that have occurred uniquely in the Oenothera plastid or mito- chondrial sequences, however, are a direct indicator of the relative number of events in these organelles since the sepa-

46

I ATCTGCCTATGAACCTTGGTTCACGCTCTACGTATCTTATTCTTTCAAGCCAAA•AAATGAATGAACCATCGAGTCGAGA

81 AGACCGAAGCGGAAGGGTCTTTTCTCA•AGCCCGGAAAGCGAAGTCGAAGCGATACGTTACAGCCTGCCG•ACGACTTTA

~6~CAT~TTTTATTG~AACTTACTTAGAAGAAATTCTTTC~CT~TTGTATCTGAT~GTAGTG~TGAAGGGTT~GGTTC~T

~ACTCTT C T TTTT GGTTT CCTTT TT TGCCTATGCTTAAGGG 241[A TCACCGGTTA CGGCTTGAA AAAGCAA GGCCTT TCTAAC C A

[ Mlu I 32•••ATGATTCCCTATGGGT•TAACCCGCTTCCATACTCATCTTCATCAGGAGGACACATAG•CT•GCTTCTAGCACG•GTAA

401

481

561

641

721

801'

881

961

1041

112]

120]

1281

1361

1441

1521

1601

1681

176]

184]

1921

200]

2081

2161

2241

2321

240]

GAGGTGCTTGATGTCCTTCCCCCCCTCAAAAGCAGCCGGAGTCGATTCTAGCTGCCTGTAATGGCTCCTCCTGGGATGGG

Nhe I Nde I •TTT•TGATGCCTTCGATGTTGCC•AGTAGTATAGTTGTGTAACTGGCTAGCTTTTA•AGTACGGAACATATGCTTGTAG

A~GGAA~AT~T~CCTGTAGCGTCCTTCCGTCAAGATGTCAAACAAAAAGTAGATCCCACT~A~CA~ATTGAGAATCTTGT[

Nhe I GTTAAGCT•TAGGGTTAGTGAG•ACCAACAAGTGTTACAGGCTAGCAAGCGAACAAGCGAGCTACGGACAAGAGTCATAC

pt 23S rRNA---~[ G OTTGT TTTCCCCC OTO OTCTGCTrT~CCOr T OCTC CTO TCO OCOCTCTTOCOCCG ~ATGAACOGG C | Nsi I

•CTAAGCGATCTGCCGAAGCT•C•GGATGTAAAAATGCATCGGTAGGGGA•CGTTCCGCCTT••AGGGAAGCACCC•C•A

GAGCGGGG•TGGACGAAGCGGAAGCGAGAATGTCGGCTTGAGTAACGCAAACATTGGTGAGAAT••AATGCCCCGAAAAC

•TAAGGGTTCCTCCGCAAGGTTCGTCCACGGAGGGTGAGTCAGGGCCTAAGATCAGG•CGAAAGGCGTAGTCGATGGACA

ACAGGTGAA~ATTCCTGTA~TACCCCTTGTTGGTCCCGAGGGACGGAGGAGGCTAGGTTAG~CGAAAGATGGTTAT~GGT

TCAAGGACGCA AGGTGCCCCTGCTTTTTCAGGGTAAGAAGGGGTAGAGA AA ATGCCCCGAGCCAATGTTCGAGTACCAGG

Kpn I TGCTACGGCGCTGAAGTAACCCATGCCATACTCCCGCTCGAACGACCTTCAACA AAAGGGTACCTGTACCCGAAACCGAC

ACAGGTGGGTAGGTAGAGAATACCTAGGGGCGCGAGACAACTCTCTCTAAGGAACTCGGCAAAATAGCCCCGTAACTTCG

Sma I GGAGAAGGGGGTCGCAGTGACCAGGCCCGGGCGACTGTTTACCAA•AACACAGGTCTCCGCAAAGTCGTAAGACCATGTA

Bat E I I TGGGGCTGACGCCTGCCCAGTGCCGGAAGGTCAAGGAAGTTGGTGACCTGATGACAGGGGAGCCGGCGACCGAAGCCCCG

GTGAACGGCGGCCGTAACTATAACGGTCCTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTC~GACCCGCACGAAAGGCGT

~ACGATCTGGGCACTGTCTCGGAGAG AGACTCGGTGAAATAGACATGTCTGTGA AGATGCGGACTACCTGCACCTGGACA

Hind I I I GAAAGACCCTATGAAGCTTTACTGTTCCCTGGGATTGGCTTTGGGCCTTTCCTGCGCACCTTAGGTGGA•GGCGAAGAAG

Apa I GCCTCCTTCCGGGGGGGCCCGAGCCATCAGTGAGATACCACTCTGGAAGAGCTAGAATTCTAACCTTGTCTCAGGACCTA

CGGGCCAAGGGACAGTCTCAGGTAGACAGTTTCTATGGGGCGTAGGCCTCCGGGTA ACGGAGGCGTGCAAAGGTTTCCTC

Xho l GGGC•GGA•GGAGATT••CC•T•GAGT•CAAAGGCAGAAGGGTGCTTGACTGCAAGAC•CAC•CGTCG•••AGGGACGAA

AGTCGGCCTTAGTGATCCGACGGTGCCGAGTGGAAGG•C•GTC••TCAA•GGATAAAAGTGACTCTAGGGATAA•AGGCT

Sst I GATCTTCCCCAAG•GCTCACATCGACG•CAAGGTTTGCCACCTCGATGTCGGCTCTTCGCCACCTGGGGCT•TAGTATGT

TCCAAGGGTTGGGCTGTTCGCCCCGGTACGTGAGCTGGGTTCAGAACGTCGTG AGACAGTTCGGTCCATATCCGGTGTGG

GCGTTAGA•CATTGAGAG•ACCTTTCCCTAGTACGA•AGGACCCGGAAGGACGCACCTCTGGTGTACC•GTTATCGTGCC

CACGGTAAACGCTGGGTAGCCAAGTGCGG AGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGT

GCTCTCC+TTCCGACTTCCCCGGAGCCTCCGGTAGCACAGCCGACACAGCGACGGGTTCTCTGCCCCTGCGGGGATGGA

47

pt 4 . s s rRN^ ~ 1 I 2481 GCGACAGAAGTTTTGCGAATTCAAGA~AgG~TCACGGC~AGACGAGCCGTTTATCATTATGATAGGT~T~AAGTGGAAGT ]

2561 [ Pvu I |

I GCAGTGATGTATGCAGCTGAGACATCCTAACAGACCGGTAGACTTGAATTTGTTCCTACATGACCCGATCAATTCGATC

2641 AGG•ATTCGCCATCTATTTTCATTGTTCAACCCTTTGA•••CATGAAAAA•CCAAAAGCT•T••CCTCCCTCTCTATCTA

2721 TCTATCCAACCAAGGGATGGAAG•GCGGAGGCCTTTGGTGTCCCCTCCAGTCAAGAATTGGGGGGGCCTCACAATGACTA

2801 GTCA•'•••TGCTTTTC•I•CTTATG(•CTTTCCTCGTCTTTGTGT•ATCTAA•GC•TGTTACGATAGGAA•AGTGG•••GTGTA

Barn I|I 2881 CTG(',CTTAG(;CAAAGGATCC D

Fig. 2. The nucleotide sequence shown of linkage group III (Fig. 1) contains the entire region of the integrated sequence from the distal portion of the plastid rRNA cistron. The borders of the plastid homology are indicated with C and D as described in the text and in Fig. 3. The coding regions within this plastid fragment are boxed by the solid line and their orientation is indicated by the arrows. The broken line encloses the 889 nucleotide region also present in linkage group II connected to part of the open reading frame potentially encoding the ribosomal protein $13 (Fig. 1)

ration of the mitochondrial and the plastid sequences in this organism.

To determine the sequence evolution rates in the differ- ent organelles we analysed the differences between the Oen- othera mitochondrial and the tobacco and Oenothera plas- rid sequences in the T-terminal region of the 23 S rRNA, the adjacent intergenic spacer and the 4.5 S rRNA regions. These divergences result from single events in one of the three sequences: two duplication events have occurred in the mitochondrial copy only, one of five adjacent nucleo- tides 5'-TCTAT-3' and one of three consecutive G residues. Four events have altered the Oenothera plastid sequence with three single nucleotide substitutions and one deletion of nine nucleotides. Thirteen differences are observed be- tween the tobacco plastid and both Oenothera plastid and mitochondrial sequences; 10 single nucleotide exchanges, 1 deletion of 4 nucleotides and 2 duplication events of 3 and 32 nucleotides, respectively. The latter duplication in the intergenic spacer region between the 4.5 S and the 5 S rRNA genes contains two nucleotide exchanges relative to the adjacent progenitor sequence.

The transfer of these sequences to the mitochondrion occurred subsequently to the divergence of Oenothera and tobacco, as the mitochondrial sequence is more closely re- lated to the Oenothera plastid sequence (6 differences) than the tobacco sequence (15 differences). In four of the six differences between the Oenothera plastid and mitochondri- al sequences, the mitochondrial sequence resembles that of the tobacco chloroplast. This suggests that these are recent changes within the Oenothera plastid, and that they have occurred since the transfer of this sequence to the mitochon- drion.

The observed independent sequence divergences, four in the Oenothera plastid sequence and two in the Oenothera mitochondrial copy, are too few for a quantitative evalua- tion of evolutionary rates. Comparisons of sequence diver- gences between mitochondrial genes of different species have however indicated comparatively slow sequence evolu- tion in plant mitochondria (Hiesel and Brennicke 1983; Schuster and Brennicke 1985, 1986).

The differences between the Oenothera plastid and mito- chondrial sequences suggest that this transfer of sequences between organelles was an ancient event that occurred once and is not an ongoing process, which would allow the two

genomes to correct the divergent drift of their two se- quences.

Analysis of integration borders

The mechanism of interorganellar sequence transfer and integration is as yet unclear and nothing is known about the requirements and specificities of the integration process. The border regions of such transferred and integrated se- quences are potentially useful sources of information on the mechanisms involved, since they might play a role in selection of excision and integration sites.

The 5' border of the plastid sequence in the Oenothera mitochondrial genome lies within the 23 S rRNA coding region of the plastid DNA (Fig. 3 a, b, border C). The 3' border (D) of the integrated fragment is defined by the end of homology in the intergenic spacer region just before the beginning of the 5 S rRNA gene in the plastid sequences (Fig. 3 a, c).

Comparison of the nucleotide sequences around the two integration borders 5' (C) and 3' (D) indicates that these two sequences are entirely unrelated (not explicitly shown, sequences are given in Figs. 2 and 3 b, c). This observation suggests that either the interorganellar transfer and integra- tion process occurs at random locations or that the mecha- nism requires more than mere common sequence motifs.

The 5' rearrangement border (C) however shows an in- triguing coincidence in its intramolecular location in the plastid large rRNA sequence with an intramitochondrial DNA reorganization event within the mitochondrial 26 S rRNA gene (Fig. 3 a). Site-specific circularisation at this lat- ter locus leads to formation of the small circular molecule #3 (Manna and Brennicke 1986). The two independent recombination sites are only 21 nucleotides apart in the alignment of the different rRNA coding regions (Fig. 3 b). It is as yet unclear whether these locations imply a common recognition mechanism, perhaps involving secondary struc- ture elements, for sequence rearrangement activity in the Oenothera mitochondrion.

Secondary intramitochondrial rearrangements

The plastid sequence in the Oenothera mitochondrial ge- nome described above has been involved in a subsequent

48

mf I

mf

mf p~

i----- -~

I

26 S r RNA i

I pseudo I

1

I t - [ 3 ' 23 S r RNA I

~+.5 Sr RNA__I 5SrRNA

23 S r RNA

t C D

mt 26S rRNA 66T66~CTT66AA6CA6CCATCCTTT~AACJAAA6C6TAATC6CTCACT66

mt pseudo 666AA666C66TT6CA6CCATCCTTT6AAGAAA6C6TAATA6CTCACT66

mtpt 23S rRNA 6A6TTQTATTTCCCCCAGT6AA6TCT6CTTTTTCC6TAATA6CTCACT6A

pt 23S rRNA 6BTTT6CCTA6AA6CA6CCACCCTTCAAA6A6T6C6TAATA6CTCACT6A ¢

b C

Oep~

Oem~

Ntpl

Oept

Oemt

Ntpt

23SrRNA C6~A6C66ATAACT(~CT~AAA6CATCTAA6TA6TAA6CC~ACCCCAA6AT6A6T(iCTCTCCl~ATTCC6ACTTCCCC66A6CCTCC(]6TA6CACA6CC(~A(i

~+.5S r RNA A~A6C6AC666TTCTCT6CCCCT6C6666AT66A6C6ACA~A6TTTT--6C6AATTCAA6A~AA66TCAC66C6A0AC6A6CC6TTTATCATTAT6AT ~~TT~T~T~T~T~A~T~TT---~TT~T~C~TTT~T~TT~T~T

Oep~ A~6T6TCAA6T~6AA6T6CA6T6AT6TAT6CA6CT6A6ACATCCTAACA~CC6CTA~ACTT6AA(

Oemt A66T6TCAA6T66AA6T6CA6T6AT6TATGCA6CTGAGACATCCTAACA6ACC66TA6ACTT6AA(

N t p ~ A66T6TCAAGT66AA~T6CAGT6AT6TAT6CA6CT6A66CATCCTAACA~ACC~6TA6ACTT6AA(

TT6TTCCTACAT~ACCC6ATC . . . . . . . . . A66

CTT6TTCCTACAT6ACCC6ATCAATTCGATCA66

;TT6TTCCTACAT6ACCT6ATCAATTC6ATCA66

O e p t CATTC6CCATC~ATTTTCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TT6TTCAACCCTTT6ACAACAT6AAAAAACCAAAA6CTCAGCCCTCCCT

Oemt CATTC6CCATCTATTTTCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TT6TTCAACCCTTT6ACAACAT6AAAAAACCAAAA6CTCT6CCCTCCCT

N ~ p t CAcTC6CCATCTATTTTCATT6TTCAAATCTTT6ACAACAC6AAAAAACCATT~TTCAACTCTTT6ACAACAT6AAAAAACCAAAA6CTCT6CcCTCCCT

O e p t CTCTATCTA . . . . CCAACCAA6G6AT66AAG6GCG6A6CCCTTT66T6TCCCCTCCA6TCAA . . . . . 6666---CCTCACAAT6ACTA6TCAATATBCT

Oemt CTCTATCTATCTATCCAACCAA666AT~6AA666C66A6~CCTTT66T6TCCcCTCCA6TCAA6AATT6666666~CTCACAAT6ACTA~TcAATAT6cT

Ntpt CTCTATCTATC . . . . . . . . CAA••6AT••AA••••A•A•GCCTTT••T•TC•CCTCCA6TCAA6AATT••6•-••CCTCACAATCACTA•CCAATAT6CT

49

Oept

Oemt Ntpt

D ,~ 5SrRNA T~T~T*C~T*~*T*T*A*1G~CT~T~T~c~T~*G*TTCA~a~GTTc(iA~ATTcTGGT~*GT*cCTAGGcGTAGA*(3*GAA~cA~cA~MT~ATc~cG*AA~TTG*GTGG~TAAACT~TA~T

TTTCTCTTATGCCTTTCCTC(iTCTTTGT~TAATCTAAG~CATGTTACGATAGGAAAAGTGC~AAGTGTAcTGGCTTAGGCAAAGGATCC TTTCTCTcAT;CCTT~CT~C;TTCAT~(~T~C(3A~TATTCT(~6T;TCCTA~GC~TA(~AG~AACCACACCAATCCATCCc;AAcTT;~T(}GTTAAACTCTACT

Oept GCO(~T(IAC(3ATACT(~TAG66(3A(~(3TCCT{~C6GAAAAATA(3CTC6AC6CCA(~(~AT

Ntpt GCGGTGACGATACTGTAGGGC~AG6TCCTGCG6AAAAATAGCTCGACGCCAGGAT C Fig. 3a-c. Analysis of the interorganellar integration borders, a Schematic comparison of the interorganellar transposition of a sequence from the plastid ribosomal cistron (pt 23 S rRNA, 4.5 S rRNA and 5 S rRNA) with the location of an intramitochondrial event involving circularisation of a small DNA molecule out of the main mitochondrial genome with a truncated copy (mt pseudo) of the mitochondrial 26 S rRNA gene (mt 26 S rRNA; Manna and Brennicke 1986). The mitochondrial copy (rot pt) of the plastid 23 S rRNA gene is a portion of linkage group III in Fig. 1. The region enclosed by dashed lines is shown in detail in b (border denoted C) and e (border denoted D). b The sequence of the site for the intramitochondrial circularisation of the mitochondrial 26 S rRNA gene with a pseudogene is compared with the 5' integration border C of the plastid sequence in the mitochondrial genome. Arrows indicate these two rearrangement sites that are separated by only 21 nucleotides in the large rRNA coding regions. Secondary structure preferences might influence site selection for these recombination events, c Nucleotide sequence comparison of the T-terminal region of the plastid 23 S rRNA, the 4.5 S rRNA and the 5 S rRNA cistrons in Oenothera (Oept) and tobacco (Ntpt; Shinozaki et al. 1986) with the integrated plastid sequence in Oenothera mitochondria (Oemt). Coding regions are indicated with solid boxes, only interrupted for the plastid 5 S rRNA which is not present in the mitochondrial genome of Oenothera. The 3' integration border D is marked with an arrow 11 nucleotides upstream of the 5 S rRNA. Nucleotide sequence differences indicating fast sequence evolution in plastids and slow sequence drift in mitochondria are discussed in the text

in t rami tochondr ia l sequence rearrangement (Fig. 1, l inkage group II) with the mi tochondr ia l gene putat ively encoding the r ibosomal protein $13 discussed below (Fig. 1, l inkage group I). In this event 889 nucleotides have been duplicated from linkage group II I into l inkage group II (Fig. 1). This new molecular ar rangement now connects the region up- stream of the cytochrome oxidase subunit II gene (COX II; Fox and Leaver 1981; Hiesel and Brennicke 1983, 1985) with the sequence upstream of the gene encoding subunit i of the N A D H dehydrogenase (Schuster and Brennicke, in prepara t ion) on opposi te strands. The duplicated region between the borders A and B has been sequenced in both alleles and found to be identical (data not shown).

The rearrangement combines the first par t of the mito- chondrial plastid 23 S r R N A sequence with the 3' por t ion of the S13 open reading frame at border B as indicated in Figs. 1, 3 and 5. This and the 5' border A of the sequence common to both alleles are shown in detail in Fig. 4 with nucleotides common to all l inkage groups at border B boxed. The relevance of these homologies is unclear, since none are found a round border A or any of the integrat ion borders of the plast id D N A fragment (borders C and D in Fig. 3).

O R F homologous to ribosomal protein S13

One of the mi tochondr ia l l inkage groups involved in the in t rami tochondr ia l sequence rearrangements described above contains the gene for subunit I of the cytochrome oxidase subunit I complex (COX I), an O R F with homolo- gy to the r ibosomal protein S13 (S13) and the locus encod- ing subunit I of the N A D H dehydrogenase (ND 1 ; l inkage group I in Fig. 1).

The sequence rearrangement occurs within the O R F with high homology to the tobacco mi tochondr ia l O R F putat ively encoding the r ibosomal protein S13 (Bland et al. 1986). I t is located downst ream from the COX I (Isaac et al.

1985; Hiesel et al. 1987) and upst ream of the N D I genes. This reading frame shows high homology with the respec- tive tobacco sequence (Fig. 5), with only 22 nucleotide ex- changes of which 6 are due to a deletion of 2 triplets in Oenothera mitochondr ia . This in-frame deletion preserves the reading frame, suggesting that this locus codes for a genuine polypept ide in higher p lant mitochondria .

Transcr ipt ion analysis (not shown) reveals a complex pat tern for this gene with transcripts between I and 5 kb in size. The major t ranscript species with homology to the S13 O R F is 3 kb in length. A number of less abundan t smaller transcripts are detectable in hybridizat ion experi- ments. This pa t te rn is similar to that reported for S13 in maize and tobacco (Bland et al. 1986) and possibly related to the complex structure of the associated N D 1 locus (Stern et al. 1986; Bland et al. 1986). In tobacco this entire locus appears to be cotranscribed with the ATPase subunit 9 gene from the p romote r upst ream of that gene.

II

III

CATTTGTTGCAACGTTCCGTT~<~CATCTTTTATT

ACAGCCTGCCGCACGACTTTA~: CATCTTTTATT

Fig. 4a, h. The intramitochondrial sequence rearrangement borders A (a) and B (b) are compared in the different linkage groups in- volved in these events. Linkage groups are numbered as in Fig. 1. The surrounding nucleotides show some homology around border B, as indicated by the unbroken boxes. No such homology is de- tected between the seemingly unrelated sequences near border 'A ' . Stippled boxes indicate identical sequences

50

~ B M S Y I S G A R S V A D E q V R A S T K M D (~ I

Oe 1 ATG TCA TAT ATT TCA G(3A GCT A~ TCA Gl-[ GCC GAT (~AA CAA 6TA AC_~ ATT GCC TCA ACA AAA ATG GAT (~C~ ATA

Nt 1 ATG TTA TAT ATT TCA (~GA GCT A~ TTA (}TT GGC GAT (]AA CAA GTA A~ ATT ~CC TCA ACC AAA ATT GAT (~c.~A Al-I M L Y I S G A R L V (3 D E Q v R I A S T K I D (3 I

5 P K K A I Q V R S R L ~ G N I P R K E L T K (~ 76 ~GA CCT ;EAA AAA ~CC ATT CA~ BTT C~T TCT C~A TIA ~T . . . . . . ~ AAC ATC CC~ AC~ AAA C~A~ TTA ACT AA~

Nt 76 ~SA CCT AAA AAA GCC ATT CAB ~T C~T TAT CGA TTA ~GT ATC A~T G8A AAC ATA AA~ ATA AAA GAA TTA ACT AAB P K K A I Q V R Y R L ~ I S G N I K I K E L T K

Y Q I D Q I E Q M R ~ Q D H V V H W E L K R ~ E R Oe 145 TAT CAG ATC C.~C CAA ATT ~ CAA ATQ A~ ~T CAA BAT CAT ~TT 8TT CAT T~8 ~AA TT~ AA~ A~ ~6A ~AA C~A

Nt 151 TAT CAA ATC ~AC CA.A ATT ~AA CAA AT~ ATA ~T CAA CAT CAT ~TT ~TT CAT TS~ ~AA TT~ AA~ A~8 ~SA GAA C~ Y Q I D Q I E Q M I ~ Q D H V V H W E L K R ~ E R

A D I E R F I S I S C Y R G I R H Q D G S P L R G Oe 220 GCA GAC ATC GAA CGA TTC ATT TCT ATT TCT TGT TAT CGT G~ ATT CGT CAT CAA C~AT G~ TCG CCC TTA CGC GGI

Nt 226 GCA 8AC ATC GAA CGA TTA ATT TC~ ATT TCT T~T TAT CBT ~GA ATT CGT CAT CAA~AT B~A TC~ CCC TTA C8C ~T A D I E R L I S I S C Y R ~ I R H Q :.D ~ S P L R G

q R S H T N A W T S R K R I W g * Oc ~ 295 CAA CC~ AGT CAT ACT A.AT GCT CGG ACT TCT CGC AAQ CGA ATT CGQ ~ TGA

Nt 301 C#.A C~ ATC CAT ACT #AT GCT AGG ACT TGT CGC ~G CTA ATT CG~3 ~ TGA Q R T H T N A R T C R K L I W K *

Fig. 5. An open reading frame with homology to the ribosomal protein S13 is found in a similar genomic location in Oenothera (linkage group I in Fig. 1) and in tobacco and maize mitochondria (Bland et al. 1986). Deletion of two triplets in the Oenothera open reading frame (Oe) in comparison with the tobacco sequence (Nt) preserves the reading frame which is otherwise highly conserved between the two species, indicative of a functional polypeptide encoded by this sequence. A truncated copy of this gene is created by intramitochon- drial sequence rearrangement at border B with the sequence of plastid origin in linkage group III of Fig. 1

In conclusion, we have described the first sequence anal- ysis of a fragment of plastid origin in the mitochondrial genome of a higher plant which has undergone subsequent further rearrangements with other mitochondrial sequences. One of these involves the gene putatively encoding the ribo- somal protein S13 and creates a fusion product of the 3'-ter- minal region of this gene with part of the plastid 23 S r R N A sequences.

This plastid sequence is derived from the rRNA cistron, a region of the plastid genome that is abundant ly tran- scribed. All other incidences of plastid or nuclear sequences in the mitochondrial genomes of higher plants so for re- ported likewise originate from transcribed regions. Intrami- tochondrial reverse transcription of imported R N A mole- cules from either plastids or nucleus could be one of the mechanisms responsible for the interorganellar transfer o f genetic information besides the hypothetical transfer of nu- cleic acid sequences in the form of D N A molecules. This model is supported by the recent definition of a reverse transcriptase-like reading frame in Oenothera mitochondria (Schuster and Brennicke 1987).

The sequence transfer described here seems to have oc- curred as a singular event since the two formerly identical Oenothera plastid genome and mitochondrial sequences have had time to diverge. The nucleotide substi tution rate in plant plastids observed here appears to be much faster than sequence drift in the plant mitochondrial genome.

Acknowledgements. We are grateful to Drs. P.G. Isaac and C.J. Leaver (University of Edinburgh) for their help with the data anal-

ysis, for first pointing out the plastid homology to us and for many discussions of the manuscript. We thank J. vom Stein and W. Hachtel for the chloroplast DNA clones established from green leaves and related unpublished data. U. Christner did the tissue culture work, V. Uhle-Schneider artwork, and C. Specht photogra- phy. The generous financial support of the Deutsche Forschungsge- meinschaft made these experiments possible. A.B. is a Heisenberg Fellow.

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C o m m u n i c a t e d by R. H e r r m a n n

Received May 12, 1987