Mitochondrial genome rearrangement leads to extension and relocation of the cytochrome c oxidase subunit I gene in sorghum

Cell, Vol. 47, 567-576, November 21, 1986, Copyright 0 1986 by Cell Press

Mitochondrial Genome Rearrangement Leads to Extension and Relocation of the Cytochrome Oxidase Subunit I Gene in Sorghum Julia Dailey-Serres: Deborah K. Hanson,t Thomas D. Fox,t and Christopher J. Leaver* * Department of Botany University of Edinburgh Edinburgh EH9 3JH Scotland tSection of Genetics and Development Cornell University Ithaca, New York 14853

Summary The mitochondrial gene (COXI) encoding cytochrome c oxidase subunit I (Cot) was isolated from two cytoplasmic genotypes of sorghum that synthesize different COI polypeptides. The Milo COI (M, ~38,000) is encoded by a 530 codon structural gene sharing 98% homology with the corresponding maize gene. A variant CO1 observed in 9E cytoplasm (M, m42,OOO)is encoded by a 631 codon structural gene that diverges completely from the Mile COXI gene both 100 bp 5’to the presumed initiator methionine and within the 3’ coding sequence. The 3’ divergence results in a 101 C-terminal extension of the 9E COI that is not homologous to any known mltochondrial polypeptide. The novel 9E COXI, apparently arising from at least two rearrangements affects transcription and gene product.

Introduction Mitochondrial genomes of ,higher plants are large and variable in size. In both maize (Zea mays, 570 kb) and brassica (Brassica campestris, 218 kb) the mitochondrial genome has been mapped as a large circular molecule (Lonsdale et al., 1984; Palmer and Shields, 1984). These studies indicate that mitochondrial genomes of higher plants have a complex and multipartite structure because of intramolecular recombination events between directly or inverted repeated DNA sequences that give rise to subgenomic molecules. Homologous recombination between repeated DNA sequences in the mitochondrial genome of S cytoplasm maize and two self-replicating linear molecules (Sl, 6.4 kb; S2, 5.4 kb) results in the formation of large linear DNA molecules with Sl or S2 attached at one end (Schardl et al., 1984). Repeated DNA sequences that may be involved in homologous recombination events have been identified near, within, and containing mitochondrial genes in a number of plant species: maize (Leaver et al., 1985; Dawson et al., 1986; Dewey et al., 1985) Brassica (Palmer and Shields, 1984), Triticum (wheat) (Falconet et at., 1984, 1985; Bonen et al., 1984), and Oenothera (evening primrose) (Manna and Brennicke, 1986). Thus, the physical complexity of plant mitochondrial DNA (mtDNA) appears to arise from genome

rearrangements involving homologous recombination between repeated DNA sequences. A mutation associated with variation in mitochondrial genome organization is cytoplasmic male sterility, the maternally inherited phenotype in which pollen development or anther dehiscence in impaired but female fertility is normal (Duvick, 1965; Edwardson, 1970). The cytoplasmic male sterile (CMS) phenotype is observed when a nucleus from one race or species is placed into a foreign cytoplasm by backcrossing. Nuclear-cytoplasmic combinations that display the CMS phenotype lack dominant fertility restorer genes (Edwardson, 1970). Thus, the CMS phenotype results from incompatibility between a nucleus and a foreign cytoplasm. Marked and characteristic differences in mitochondrial genome organization, as detected by restriction endonuclease digestion of mtDNA, are observed between normal and CMS-conferring cytoplasms of maize, sorghum (Sorghum bicolor) and other plant species (Levings and Pring, 1976; Pring and Levings, 1978; Ring et al., 1982; Mikami et al., 1964; Boeshore et al., 1985). In addition, characteristic variant polypeptides are synthesized by mitochondria isolated from male sterile nuclear-cytoplasmic combinations (Forde et al., 1978; Dixon and Leaver, 1982; Boutry and Briquet, 1983). While examining the polypeptides synthesized by isoPated mitochondria from various sorghum lines, we found that mitochondria from CMS lines containing 9E cytoplasm synthesize a variant form of cytochrome c oxidase subunit I (COI) (Dixon and Leaver, 1982). Thisvariant polypeptide has an apparent molecular weight of 42,000 (42 kd), as estimated by SDS-polyacrylamide gel electrophoresis, and replaces a normal 38,000 dalton (38 kd) COI polypeptide synthesized by Milo and other sorghum cytoplasms (Dixon and Leaver, 1982). In this paper we provide evidence that synthesis of the variant COI by 9E cytoplasm mitochondria is due to a mutation in the coding region of the gene and is not the result of an alteration in RNA processing, translation, or posttranslational processing. We have isolated the cytochrome c oxidase subunit I gene (COXI) from the mtDNA of a line that synthesizes the normal 38 kd form of COI (Kafir nucleus in Milo cytoplasm, referred to as Milo) and from 9E cytoplasm (Kafir nucleus in gE cytoplasm, referred to as 9E). Sequence analysis revealed that the 38 kd and 42 kd forms of COI are encoded by two different genes. Genome rearrangements have occurred in 9E mtDNA both 5’to the initiator methionine and within the 3’ coding region of the COXI gene. These events have resulted in the 3’ continuation of the COXI open reading frame (ORF), alterations in gene transcription, and an increase in the molecular weight of the CO! polypeptide. Results Identification of the Normal and Variant Forms of Cytochrome c Oxidase Subunit I To confirm that the variant 42 kd form of CO1 is synthesized in vivo as well as by isolated mitochon

Celi 566

IO. 9

Figure 1. identification of the Normal and Variant Cytochrome dase Subunit I (COI) Polypeptide in Sorghum

c Qxi-

(Lanes A and B) lmmunoprecipitation of COI from Lj3sS]methioninelabeled sorghum mitochondrial translation products. Mitochondrial polypeptides were immunoprecipitated with an antiserum against yeast COI, fractionated by electrophoresis on a 16% (w/v) SDSpolyacrylamide gel, and fluorographed. lmmunoprecipitate from Milo mitochondria (lane A) and 9E mitochondria (lane B). (Lanes C and D) Sorghum mitochondrial polypeptides immunolabeled with an antiserum against yeast COI. Mitochondrial protein (100 ug) was fractionated by electrophoresis in each lane of a 16% (w/v) SDS-polyacrylamide gel. The polypeptides were immobilized on nitrocellulose by electrophoretic transfer, incubated with antiserum prepared against yeast COI, washed, and labeled with rz51-protein A by the method of Batteiger et al. (1982). Milo mitochondrial protein (lane C), 9E mitochondrial protein lane D). 9E mitochondrial protein reproducibly gave a weaker signal in this reaction.

a rabbit antiserum prepared against yeast COI to identify the homologous protein in sorghum (Dixon and Leaver, 1982). Mitochondria were isolated from Milo and 9E cytoplasms and were allowed to synthesize protein in the presence of L-[ssS]methionine (Leaver et al., 1983). The subunit I polypeptide was identified by immunoprecipitation of labeled mitochondrial translation products (Figure 1, lanes A and B). As shown previously, mitochondria isolated from Milo synthesized a COI polypeptide with an estimated molecular weight of 38 kd. 9E mitochondria synthesized a subunit I polypeptide with an apparent molecular weight of 42 kd. To rule out the possibility that the larger form of the COI polypeptide was an artifact of the organellar protein synthesis system, COI was identified by immunological detection of nonradioactive mitochondrial proteins bound to nitrocellulose (Batteiger et al., 1982). Antiserum prepared against yeast COI identified a 38 kd polypeptide in Milo and a 42 kd polypeptide in 9E (Figure 1, lanes C and D). Therefore, the variant 42 kd form of COI appears to be a structural component of cytochrome c oxidase enzyme complex in 9E mitochondria. Further evidence that the 42 kd COI is part of a functional cytochrome c oxidase is provided by the observation that the cyanide-sensitive oxidation of cytochrome c by mitochondria isolated from 9E and Milo seedlings were indistinguishable (Dixon and Leaver, 1982). In addition, the spectra of reduced mitochondrial cytochromes at liquid nitrogen temperature were similar for Milo and 9E cyto-

A

5

F

Figure 2. Identification of the EcoRl Fragment of MtDNA Containing the Gene for Cytochrome c Oxidase Subunit I (COXI) in Sorghum MtDNA (3 pg) from Milo (lane A) and 9E (lane B) was digested with EcoRI, fractionated by electrophoresis in a 0.8% (w/v) agarose gel, stained with EtBr, and photographed. The mtDNA was transferred to nitrocellulose, hybridized with a 32P-labeled clone containing an internal portion of the maize COXI gene, and autoradiographed. A 4.3 kb fragment in Milo (lane C) and a 10.4 kb fragment in 9E (lane D) mtDNA was identified. The blot was then washed and reprobed with s2Plabeled pS9E10.4. Additional 9.5 and 2.0 kb fragments hybridized to Milo mtDNA (lane E) and a 2.0 kb in 9E mtDNA (lane F).

plasm (data not shown). We surveyed 23 different sorghum cytoplasms to determine whether the synthesis of the variant 42 kd COI was unique to CMS lines containing 9E cytoplasm. We found that the 42 kd COI replaces the normal COI in male fertile and CMS lines containing 9E or the related IS792OC cytoplasm (Bailey-Serres et al., submitted). Cloning of the Genes Encoding the Two Forms of Cytochrome c Oxidase Subunit li In an attempt to determine the molecular basis for the existence of two forms of COI in sorghum, the EcoRIgenerated restriction fragments containing the COXI gene from Milo and 9E mtDNA were identified and cloned. MtDNA was isolated from Milo and 9E seedling mitochondria, digested with EcoRl (Figure 2, lanes A and B), and probed with a 32P-labeled DNA fragment containing a portion of the maize COXI gene (Isaac et al., 1985). A

Cytochrome 569

c Oxidase Subunit

I Genes

in Sorghum

3*P-labeled pS9E10.4. In addition to the 4.3 kb fragment identified above, a 9.5 kb EcoRl fragment in Milo mtDNA hybridized strongly to this 9E fragment (Figure 2, lanes E and F). Four additional EcoRl fragments (4.1,2.9,2.0, and 1.5 kb) from both Milo and 9E showed weak hybridization to pS9E10.4 upon longer exposure of the autoradiograph (data not shown). The nitrocellulose blot was then washed and probed with the Milo pSM4.3 probe. This clone hybridized to the Milo (4.3 kb) and 9E (10.4 kb) EcoRl fragments that contain COXI and to a lesser extent to a 2.9 kb EcoRl fragment (data not shown). The weakly hybridizing bands could represent homologous mtDNA sequences present in substoichiometric amounts, regions of partial homology, or homologous sequences in contaminating nuclear or chloroplast DNA. 5

pS9E

E

IO.4

Kb

E

4

??

E .I

;

pSfl4.3

Kb

_

1 Kbu

1.75 Kb

Heteroduplex Figure 3. Heteroduplex Analysis of the (pSM4.3) kb EcoRl Fragments Containing

10.4 (pS9E10.4) and 4.3 the COXf Gene from Milo

and 9E MtDNA The cloned DNAs were digested with EcoRl to separate the cloned insert from vector DNA, subjected to denaturation and renaturation, and then prepared for electron microscopy by the formamide-protein monolayer technique of Davis et al. (1971). (A) Electron micrograph of a representative heteroduplex molecule (n = 15). The 1.75 kb (SD * 3.09 kb) heteroduplex region is shown. M13, single-stranded DNA marker (7.13 kb); PAT, double-stranded DNA marker (3.6 kb). (B) Diagrammatic representation of the heteroduplex, showing the location of COXI on each mtDNA fragment. (E, EcoRI).

kb EcoRf-generated mtDNA fragment from Milo and a 10.4 kb EcoRl-generated mtDNA fragment from 9E hybridized to the probe (Figure 2, lanes C and D). We have found that the gene is located on a 4.3 kb EcoRI-generated fragment in all lines that synthesize the 38 kd form of COI, and on a 10.4 kb fragment in all lines that synthesize the 42 kd form of COI (data not shown). The 4.3 kb EcoRl fragment from Milo mtDNA was cloned into pBR325 (Bolivar, 1978) and the clone was designated pSM4.3. The 10.4 kb 9E mtDNA fragment was cloned into the plasmid vector pNS1 (Nikolnikov et al., 1984) and the clone was designated pS9ElO.4. 4.3

Heterodupiex Analysis of the CLoned Genes DNA heteroduplex analysis was performed to determine the extent of sequence homology between the mtDNA fragments in pSM4.3 and pS9E10.4 (Davis et al., 1971). A 1.75 kb (SD & 0.09 kb) heteroduplex region was identified (Figure 3A), which indicates that the two COXI genes share a 1.75 kb region and are surrounded by nonhomologous sequences (Figure 3B). Detection of SE-Specific COXI Flanking Sequences Elsewhere in the Mile Mitochondrial Genome To determine whether the nonhomologous sequences of pS9E10.4 are located elsewhere in Milo mtDNA, the DNA blot shown in Figure 2 (lanes A and B) was reprobed with

DNA Sequence Analysis of Mile COXI and Comparison with Other Known COXI Genes The Milo COXI clone pSM4.3 was digested with hexanucleotide-recognizing restriction enzymes, and restriction sites were mapped (Figure 4A). Sau3A, Mspl, and Aluf restriction fragments from pSM4.3 were subcloned into M13. Subclones that showed homology to the maize CCXI gene were subjected to dideoxy chain-termination sequence analysis (Sanger et al., 1980). The Milo gene, encoding the 38 kd form of COI, contains an uninterrupted ORF of 530 codons (1590 bp) and is similar in size to the 528 codon (1584 bp) COXI gene in maize (Table 1) (Isaac et al., 1985). The nucleotide sequence of the Milo COXI gene shows 98% homology to maize COXI and shares amino acid homology with COI in yeast (60%) Bonitz et al., 1980), Neurospora (62%~) (Burger et al., 1982), man (68%) (Anderson et al., 1981), and Drosophila (65%) (de Bruijn, 1983). The Milo and maize COXI gene sequences are essentially identical from a position -49 bp upstream of the initiation codon, through the ORF to position cl566 (Figures 48 and 4C). However, between position -16 to -21 bp the sorghum COXI gene has an additional 4 bp. This insertion in sorghum (or deletion in maize) is at the position of the putative ribosome binding site in maize (Dawson et al., 1984) (Figure 48). The first 522 amino acids encoded by the two COXI genes are identical. Homology between the Milo and maize COXI genes ends within the coding sequence, 8 and 6 amino acids (24 and 18 bp), respectively, upstream of the termination codon (Figure 4C). DNA Sequence Analysis of the 9E CQXI Comparison with the Milo Gene Heteroduplex analysis showed that a 1.75 kb region of homology exists between the COXI containing EcoRl clones of Milo and 9E mtDNA (Figure 3). A 2.2 kb BamHI-Pstl fragment from pS9E10.4 that contained the heteroduplex region and showed homology to the maize CCXI gene was subcloned into Ml3mplO (Figure 4A). This clone, designated BP2.2, was digested with Sau3A or Mspl, subcloned into M13, and sequenced. Additional Ml3 clones were made containing the Kpnl-Sau3A fragment at the 3’ end of 9E COXI. The sequence 5’to the 9E CCXI ORF and at the 3’end of the COXI gene is shown (Figures 4B and 4C).

Cell 570

Figure 4. Location, Sequence, and Rearrangementof the CQXIGene in Mile and 9E MtDNA (A)Restrictionmapof the Milo4.3 kb (pSM4.3) and 9E 10.4 kb (pSSEI0.4) EcoRlfragments,10.. cation of COXI,and restrictionmap d the re9E EBgB

*

KPH

Bg

Bg HB

H

An ORF homologous to the first 526 codons of the Mile COXI gene, but encoding a significantly larger polypeptide was identified. The presumptive COXI ORF from 9E cytoplasm is 631 codons in length and encodes a protein with a predicted molecular weight of 70,358 (Table 1). Homology between the Milo and 9E COXI genes begins at position -100 bp from the presumptive AUG initiation codon and extends to position +1579 bp within the protein coding sequence (Figure 4). There is no detectable sequence homology between the two genes 5’to position -100 bp or 3’ to position +1579 bp (within the 527th codon). Termination of the ORF encoding the 38 kd form of COI occurs 11 bp (4 amino acids) beyond the 3’point of divergence. In contrast, the ORF encoding the 42 kd form of COI continues for 314 bp more (105 amino acids). Therefore, the presumptive COI of 9E is 101 amino acids, or 12 kd larger than the corresponding protein in Milo cytoplasm. (The discrepancy between the apparent molecular weight on SDS-polyacrylamide gels, 38 kd and 42 kd, and the size predicted from DNA sequence analysis, 58 kd and 70 kd, is discussed below.) The 9E COXI coding sequence downstream of position +1579 bp bears no obvious homology to any known portion of the maize,

X

HBBg

HE

gion sequenced (E, EcoRI; B, BamHI; I-4, Hindlll; Bg, Bglll; X, Xhol; P, Pstl; S, Sau3A; M, Mspl; A, Alul; K, Kpnl). There are no sites for Sall within the 10.4 kb fragment, or for Salt, Pstl, or Xhol within the 4.3 kb fragment. (B and 6). Selected DNA and amino acid sequence of COXI from Milo (M) and 9E mtDNA. The entire COXI gene sequence will be entered into the EMBL and Genbank databases. Numbering of the nucleotide sequence begins at the A of the putative initiator methionine codon. The entire COXI gene sequence of Mile and 9E is not shown here because the ORF encoding COI of sorghum is virtually identical with the corresponding gene of maize (Isaac et al., 1965) (In sorghum the sequence GAAA is inserted between position -16 to -17 bp of maize.) Six conservative nucleotide differences between positions +1 and +I566 bp were identified (maize to sorghum): position 426 bp, T to C; 961 bp, T to A; 993 bp A to T; 1374 bp, C to T; 1551 bp A to T; 1657 bp C to A. The 5’(-49 bp) and 3’ (+1566 bp) points of divergence between sorghum and maize are indicated. The 5’ (-100 bp) and 3’ (+I579 bp) points of divergence between Milo and 9E are indicated. An 6 bp repeated DNA sequence at the 5’ divergence point, a 10 bp repeated sequence in Milo, and a 10 bp + 16 bp repeated sequence in 9E at the 3’divergence point are boxed. The Sends of the major RNA transcripts at position -404 bp (Site I) in Milo and at position -51 bp (Site II) in Milo and 9E are indicated, the putative ribosome binding site (RBS) is underlined, and a 14 bp inverted repeat located just 3’to the Milo CQXI stop codon is indicated by two arrows.

wheat, yeast, Aspergillus, or human mitochondrial genomes or any sequences in the EMBL (Release 6) and Genbank databases. However, by using a 32P-labeled DNA probe from the unique portion of the 9E COXl gene, we have determined that a homologous sequence is located elsewhere in Milo and 9E mtDNA (data not shown). Examination of codon usage in the 3’extension of the 9E COXI gene, from the divergence point at position +1579 bp to its termination at position +1893 bp, reveals significant differences from the codon usage in the Milo COXI gene. The codon CGG occurs three times in the unique portion of the 9E COXI ORF and only once in the Milo COXI gene. This codon may encode tryptophan in plant mitochondria and not arginine (Fox and Leaver, 1981). In addition, in the Milo COXI gene there is a bias for T in the third position, whereas the bias is for G in the 3’extension of the 9E COXI gene, although the G + C content of these regions is similar. Identification of COXI Transcripts Hybridization analysis confirmed that both forms of 60X1 are transcribed. MtRNA was isolated from Milo and 9E cytoplasms, fractionated by electrophoresis, blotted to

Cytochrome 571

c Cxidase Subunit

I Genes

in Sorghum

AGCT

Asw scn

1234

9E

Milo

“-3.4 2.3* 1.8. 8

A

A

E

-2.2

-24

C

B

s

SA

c

e- -2.2

5-d

D E

F

S

3’

0.2

Kb

H

r

.190f6 4186f6

Figure 5. COXf Transcript Analysis from Milo and 9E and Hybridization of Specific 3zP-Labeled COXI Clones to Milo and 9E mtRNA Mitochondrial RNA prepared from seedling mitochondria was fractionated (IO ug) under partially denaturing conditions in a 1.3% (w/v) agarose-formaldehyde gel, transferred to nitrocellulose, and probed with a 32P-labeled Ml3 DNA probe containing an internal portion of the Milo COXf gene (lanes A and B). (lane A) Milo mtRNA; (lane 8) 9E mtRNA. Parallel lanes of Milo and 9E mtRNA were transferred to nitrocellulose, cut into strips, and hybridized to specific Ml3 DNA probes (lanes C, 3, E, and F). The origin of each probe is shown in the line diagram of the Milo and 9E COXI genes below.

5.

3.4

1.8, 2.4, 2.6 Kb

-404bp site I

-5lbp srte II 540+5bp\ -

c. nitrocellulcse, and probed with a 32P-labeled Ml3 clone containing a common portion of the COXI gene (Figure 5). A major Milo COXI transcript of 1.8 kb and three minor transcripts (3.4, 2.6, and 2.3 kb) were identified (Figure 5, lane A). In contrast, in 9E a major COXI transcript of 2.2 kb was observed (Figure 5, lane B). Two 9E COXI-specific transcripts (3.3 and 3.2 kb) present in extremely low abundance were also detected after extensive exposure of the autoradiograph (not shown). We examined numerous sorghum lines and found that when the gene is located on a 4.3 kb EcoRI-generated mtDNA fragment the transcription pattern is identical with that of Mile, and the apparent molecular weight of COI is 38 kd (data not shown). In contrast, lines that contain the COXI gene on a 10.4 kb EcoRl fragment synthesize a major 2.2 kb transcript and the 42 kd form of COI. Apparently, the pattern of transcription is determined by the nonhomologous sequences flanking the Milo and 9E COXI

186+6bp’

Protected

Fragmenls

2.2 Kb

-51 bp s

(90 + 6 bp

330 bp s

401 bD

-

Protected -

Input DMA

Fragment 0.2 Kb -

Figure 6. St Nuclease Transcript Mapping of the 5’ End of the COXI Transcripts from Milo and 9E Mitochondria (A) Milo COXI: hybrids were formed with a uniformly, 32P-labeied 810 bp Mspl fragment(B) and Milo mtRNA. Hybrids ware treated with 250 unitdml Sl nuclease, and the protected hybrids ware fractionated in an 8% (w/v) polyacrylamide, 8.3 M urea DNA sequencing gel. The fragments generated were approximately 548 I 5 (fragment sized on a gel inot shown) and 186 I 6 bp (lane 1). The B’termini of these fragments correspond to positions -404 bp (Site I) and -51 bp (Site II), respectively. (B) 9E COXI: hybrids were formed with a 401 bp, 32P-labeled fragment (containing the 330 bp Sau3A fragment located at the Yend of the 9E COXI gene, C) and 9E mtRNA, digested with 250 units/ml Sl nuclease and fractionated by gel electrophoresis. Fragments of approximately 190 f 6 bp were generated (C). In addition, fragments of 330 bp (the size of the cloned insert) and 481 bp (the size of the input DNA) were observed (lane 2). As a control reaction, the Milo (lane 3) or 9E (lane 4) input DNA was hybridized to tRNA and digested with Sl nuciease.

Cell 572

genes. In order to understand the importance of gene environment on COXI expression, we have determined the 5’termini of the Milo and 9E COXI transcripts.

fished results), it remains unknown whether these ends are produced by transcript initiation or processing. Discussion

Mapping of the 5’ Termini of COXI Transcripts from Mile and 9E Mitochondria To map the origins of the multiple COXI transcripts in Mile, duplicate RNA blots were probed with specific 32P-labeled Ml3 clones (Figure 5, lanes C, D, and E). This analysis indicated that the 5’end of the minor 3.4 kb transcript begins within the Alul fragment located between position -531 and -218 bp (Figure 5, lane D). The 2.6, 2.3, and 1.8 kb transcripts hybridize to the probe used in lane E of Figure 5, and therefore their 5’ ends begin within the Sau3A fragment located from position -235 bp to +139 bp. To define the 5’ends of the Milo COXI transcripts more precisely, Sl nuclease transcript analysis was performed using a uniformly 32P-labeled, 810 bp EcoRI-Mspl fragment located from position -675 to 135 bp, which spans these S’ends (Figure 6B). The DNA fragment was hybridized to mtDNA, treated with Sl nuclease, and the protected hybrids were analyzed by electrophoresis on an 8% (w/v) polyacrylamide 8.3 M urea gel. Sl nuclease protected fragments of 540 f 5 bp (Site I) and 186 f 6 bp (Site II) were identified (Figure 6A). These termini are estimated to be -404 bp and -51 bp upstream of the presumptive AUG initiation codon (Figure 6 and Figure 4). From the transcript analysis above, we can predict that the 5’end of the 3.4 kb transcript is at position -404 bp. Since only one Sl nuclease-sensitive site is found 3’to position -235 bp, we predict that the Sl nuclease protected fragments extending to position -51 bp correspond to the 5’ termini of the 2.6, 2.3, and 1.8 kb transcripts (Figure 6B). The varied lengths of these transcripts may arise from multiple transcript termination or 3’end processing sites. The 2.2 kb COXI transcript of 9E hybridizes to a 330 bp Sau3Afragment extending from position -191 bp to +139 bp (Figure 5, lane F), and not to a fragment located upstream of this position (data not shown). The 5’terminus of the 9E COXI transcript was determined by Sl nuclease protection using a uniformly e2P-labeled 401 bp fragment containing the 330 bp Sau3A fragment (Figure 6C). An Sl nuclease protected fragment of approximately 190 f 6 bp was generated (Figure 6A), which corresponds to a S’transcript terminus at position -51 bp, within the region common to both the Milo and 9E COXI genes (Figure 6C). Hybridization of a 32P-labeled Ml3 clone specific to the 3 novel portion of 9E COXI to 9E mtRNA revealed that the 2.2 kb transcript includes the nonhomologous portion of this gene (data not shown). Thus, termination of the 2.2 kb transcript occurs downstream of the predicted stop codon, within the nonhomologous gene region (Figure 6C). Sorghum mtRNA was labeled with guanylyltransferase from vaccinia virions, which under the same conditions labels S’diphosphate termini of fungal mitochondrial transcripts &evens et al., 1981; unpublished results), in an attempt to determine whether the COXI transcripts initiate at their 5’termini. Since no labeling was observed (unpub-

Two Forms of Cytochrome c Oxidase Subunit I in Sorghum Mitochondria The cytochrome c oxidase gene in Milo cytoplasm is located on a 4.3 kb EcoRl fragment of mtDNA and encodes a 530 amino acid polypeptide (58,484 daltons), which correlates with the synthesis of the normal COI polypeptide (apparent molecular weight 38 kd). The COXI gene in Mile sorghum and maize share 98% sequence homology and encode a polypeptide of similar length (Table 1). In contrast, in SE sorghum the COXI gene is located on a 10.4 kb EcoRl fragment of mtDNA and encodes a 631 amino acid protein (70,358 daltons), which correlates with the synthesis of a variant COI polypeptide (apparent molecular weight 42 kd). Sequence analysis revealed that a rearrangement within the COXI gene of 9E cytoplasm results in the continuation of the ORF and the addition of 101 amino acids to the carboxyl terminus of the protein. The two sorghum COXI genes are identical from position -100 bp 5’to the presumptive AUG codon, to position +1579 bp within the COXI ORF (Figure 4). Directly repeated DNA sequences are located at both the 5’ and 3’ points of divergence. An 8 bp direct repeat is found within the homologous region, 3 bp from the 5’divergence point (Figure 4B). A 10 bp direct repeat is found within the COXl ORF at the 3’point of divergence. This repeat is actually part of a 26 bp direct repeat in 9E cytoplasm, since the 16 bp that follow the 10 bp are repeated in the nonhomologous region (Figure 4C). Additional fragments of Milo and 9E mtDNA have been identified that hybridize weakly to the unique 5’and 3’COXI sequences (data not shown). By characterizing these mtDNA fragments, we may be able to model the events that led to the relocation of the 9E COXI ORF Since the COXI gene is situated in a single genomic location in 9E mtDNA, we predict that the 9E COXI gene resulted from novel mtDNA rearrangements that were subsequently propagated. Such events may underlie the variation in mitochondrial genome organization observed within plant species. Our observation of rearrangement within and adjacent to a mitochondrial gene is paralleled in other organisms. In S. cerevisiae, recombination within the mitochondrial gene for cytochrome c oxidase subunit Ill has been observed (Mueller et al., 1984). In this case, a larger form of the protein, with an amino-terminal extension, was generated by recombination with another mitochondrial ORE Multiple recombination events in the mtDNA of T cytoplasm maize resulted in the formation of a novel, expressed gene that is composed of flanking sequences and/or coding regions of a number of genes of mitochondrial and chloroplast origin (Dewey et al., 1986). Evidence of mtDNA rearrangements that give rise to multiple or incomplete copies of rRNA genes have been reported in wheat and Oenothera (Falconet et al., 1984, 1985; Manna and Brennicke, 1986). In S cytoplasm maize a mitochondrial genome rearrangement between the linear S plas-

Cytochrome 573

G Oxidase Subunit

Table 1. Comparison

I Genes

of the Cytochrome

in Sorghum

c Oxidase Subunit

I Gene

and Protein from Various Organisms Protein Size from from DNA Sequence

Protein Size Estimated from SDS-PAGE (kd)

Organism

OPF (bp)

ORF (predicted amino acids)

Sorghum Milo 9E

1590 1893

530 631

58.5 70.3

Z. mays 837 N

1504

528

58.2

38

S. cerevisiae

1530

510

55.9

40

(kd)

38 42

N. crassa

1665

555

51.0

40

Bovine

1542

514

57.0

42

References

are as follows: Isaac et al., 1985; Dixon and Leaver, 1982; Bonitz et al., 1980; Burger et al., 1982; Anderson et al., 1982; Azzi, 1980.

mids and a sequence 5’to COXI appears to result in multiple genomic locations for this gene and linearization of the mitochondrjal chromosomes (Leaver et al., 1985; Schardl et al., 1985). This event occurs 5’to the presumed start of transcription and has no apparent effect on expression of COXI. Mitochondrial genome rearrangement events occur frequently as a consequence of somatic cell fusion in a number of plant species: Petunia (Boeshore et al., 1983), Nicotiana (tobacco) (Belliard et al., 1979; Nagyet al., 1981; Galun et al., 1982), and Brassica (Chetrit et al., 1985). In at least one instance a novel mitochondrial genotype arises by intergenomic recombination between parental mtDNAs (Rothenberg et al., 1985). Recent evidence suggests that specific mtDNA rearrangements in a somatic Petunia cell hybrid are linked with the expression of the CMS phenotype (Boeshore et al., 1985). Genome Rearrangement Results in Altered Transcription of COXI The genome rearrangements around the COXI ORF result in altered transcription of the 9E COXI gene. A major 1.8 kb transcript is detected when COXI is located on a 4.3 kb EcoRl fragment. In contrast, a major 2.2 kb transcript is observed when the COXI gene is located on a 10.4 kb EcoRl fragment. The 5’ends of the major transcript from both the Milo and 9E COXI genes map to position -51 bp within the 5’ homologous region (Figure 4 and Figure 6). The 5’terminus of the major maize COXI transcript (position -57 bp) is near the site mapped in sorghum. A potential 26 bp stem-loop structure that may have a role in transcript processing is found at this Sl nuclease sensitive site in sorghum and maize (Figure 7). Potential stem-loop structures are associated with the 5’ termini of the smaller, more abundant transcript of the chloroplast gene encoding the large subunit of ribulose-1,5bisphosphate carboxylase frbd.) in maize (Erion, 1985). However, in this model the end of the major transcript is at the 3’end of the stem-loop structure and not at the 5’ end as for the sorghum and maize COXI transcripts. It is not yet known whether the 5’ transcript termini at position -51 bp common to both genes, or -404 bp (Site I) unique to Milo, are generated by transcript initiation. Transcript mapping data revealed that the Milo COXl

ecu C

C

C

II

G=C G-U

U-G G A A= U u= A U-A

c=c CaG MAIZEDIYERGENC6x-“,f

;

-61 ?? 6bp**C SITE II

U

5'------- UVCCA' Figure 7. Possible Secondary Sorghum and Maize

C*G U= AS-t¶bp

5' \-AATCA-----~M]~,

Structure

around Position

-51

bp in

Computer prediction of a 26 bp stem-loop structure that could form 5’ to the COXI ORF in Milo, 9E, and maize mtDNA. This structure is located near the terminus of the major COXI transcript (position -51 bp in Milo (Site II) and 9E, or -57 bp in maize).

gene transcripts appear to end at four positions downstream of the termination codon. Termination of the major 9E transcript occurs downstream of the predicted stop codon, in the region that shares no homologies to the Milo COXI gene. The Variant COI in 9E Cytoplasm Mitoc Genome rearrangement within the COXI gene resulted in the addition of 101 amino acids at the carboxyl terminus of the 9E COI polypeptide. Thus, the predicted 9E COXI gene product is 12 kd larger than in Mile, and the largest identified to date (Table 1). Apparently the 42 kd form of CO1 results from the expression of this variant gene. The molecular weight of COI, as estimated by SDS-polyacrylamide gel electrophoresis, normally differs from the molecular weight calculated from the gene sequence (Table 1). This discrepancy is most likely due to the anomalous mobility of COI in SDS-polyacrylamide gels, as observed for other hydrophobic mitochondrial proteins, inciuding COII, where a single amino acid variation results in a change in the apparent molecular weight by several kilodaltons (Cabral et al., 1978), 66111 (Thalenfeld and

Cell 574

Tzagoloff, 1980), and apocytochrome b (Norbrega and Tzagoloff, 1980). COI is a very hydrophobic protein that may have up to 12 transmembranous segments with the carboxyl terminus of the protein located on the matrix side of the inner mitochondrial membrane (Wikstrom et al., 1985). Kyte and Doolittle’s (1982) analysis of the hydrophobicity of the 101 amino acid 3’extension of the 9E COXl ORF reveals two additional hydrophobic domains, followed by a hydrophilic region. Our data suggest that the cytochrome c oxidase activity of seedling mitochondria is not affected by this terminal extension of COI (Dixon and Leaver, 1982; and data not shown). However, the effect of this extension on the structure and function of the cytochrome c oxidase holoenzyme at other developmental stages (i.e., during pollen development) is not known. Our observations provide a specific example of the way in which mitochondrial genome rearrangements can lead to altered gene expression. Genome rearrangements associated with the 9E COXI gene lead to altered transcription and the synthesis of a significantly larger COI polypeptide. It is not known whether this alteration has any functional consequences. However, it is possible that relocation of the 9E COXI gene could affect regulation of gene expression and mitochondrial biogenesis or function during development, and in turn could interfere with the formation and release of functional pollen. The two male fertile, wild-type sorghum lines that synthesize the variant COI may possess nuclear genes that have coevolved with the mitochondrial genome rearrangements and compensate for the variant polypeptide. These genes may include the fertility ‘restoring’ genes that are absent in nuclear-cytoplasmic combinations that express the CMS phenotype. Experimental

gels containing 20 mM sodium acetate, 40 mfvl kis-HCI (pf-i 8.0), 1 mM EDTA or 89 mM Tris-borate (pH 8.0), and 20 mM EDTA. DNA was transferred to nitrocellulose (Southern, 1975) and probed with thermally denatured, radioactive DNA probes (Rigby et al., 1977; Messing, 19639. High stringency DNA-DNA hybridizations were carried out overnight at 65OC in hybridization buffer: 0.75 M NaCI, 75 mM Na-citrate (5x SSC), 50 mM NaP04 (pH ZO), 0.1% (w/v) SDS, 0.2% (w/v) bovine serum albumin, 0.2% (w/v) Ficoll (type 400, Sigma), 0.2% (w/v) polyvinyl pyrrolidone (MW 40,000), and 200 pglml sonicated, denatured herring sperm DNA (Serva Fine Biochemical@. Filters were prehybridized for at least 30 min prior to the injection of 32P-iabeled DNA. Filters were washed at 65°C (2 x 15 min) in 2x SSC, 0.1% (w/v) SDS, and (2 x 15 min) in 0.1x SSC, 0.1% (w/v) SDS. Cloning of the Mile and 9E COXl Genes A clone containing an internal portion of the maize COXI gene (gift of R G. Isaac) and the yeast strain p- DS61A422 containing a portion of the yeast oxi (COXI) gene (gift of A. Tzagoloff) were used to identify the Milo COXI gene. 32P-labeled probes were hybridized to EcoRIdigested Milo mtDNA under low stringency conditions (50°C) in hybridization buffer, and a 4.3 kb fragment was identified. Mile mtDNA (10 fig) was digested with EcoRI, fractionated by agarose gel electrophoresis; the 4.3 kb fragment was eluted and ligated into the EcoRl site of pBR325 (Maniatis et al., 1982; Bolivar, 1978). Competent E. coli Hi3101 cells were transformed with ligated plasmid DNA and ampicillin-resistant, chloramphenicol-sensitive colonies were selected. DNA was prepared (Birnboim and Daly, 1979), and a clone (pSM4.3) containing a 4.3 kb EcoRl fragment that hybridized to the yeast and maize probes was identified. A recombinant DNA library of EcoRl-digested QE mtDNA was constructed in the plasmid pNS1 (Nikolnikov et al., 1984), in E. coli HBIOI cells. Filter replicas and a permanent coilection of the recombinant DNA plasmids were made (Gergen et al., 1979). Filter replicas were probed with 32P-labeied Ml3 DNA clone containing an internal portion of the Milo COXI gene. Plasmid DNA was prepared from tetracyclineand ampicillin-resistant colonies that showed positive hybridization, and a clone (pS9E10.4) containing a 10.4 kb EcoRl fragment was identified. Heteroduplex Analysis Heteroduplex analysis was performed as described by Davis et al. (1971). Plasmid DNA (pSM4.3 and pS9E10.4) was digested with EcoRl prior to hybridization.

Procedures

Sorghum Lines Seeds of sorghum (S. bicolor; L. Moench) were obtained from Gene Dalton of Pioneer Hibred International (Plainview, TX), Dr. Keith Schertz of USDA ARS, Texas A&M University, and Dr. Orin Webster, University of Arizona. Sorghum lines were identified by nuclear and cytoplasmic genotype and fertility status. fsolation of Mitochondria, Mitochondrial DNA, and RNA Mitochondria were isolated from 5 day old dark grown sorghum coleoptiles by differential centrifugation and were further purified on sucrose gradients, as described previously (Leaver et al., 1983; Dixon and Leaver, 1982). Mitochondria were used for organellar protein synthesis (Leaver et al., 1983), isolation of mtDNA, and extraction of mitochondrial nucleic acids (Dawson et al., 1984). MtDNA was removed from total mitochondrial nucleic acids by digestion with RNAase free DNAase 1(Promega Biotec, P & S Biochemicals) 1 U/pg for 30 min at 3PC. Immunological Identification of Cytochrome c Oxidase Subunit I Organellar protein synthesis and fractionation of mitochondrial protein was carried out as described previously (Leaver et al., 1983). COI was identified by immunoprecipitation (Dixon and Leaver, 1982) and immunoblotting (Batteinger et al., 1982) with a rabbit antiserum raised against yeast COI, obtained from G. Schatz. DNA Digestion, Electrophoresfs, Transfer to Nitrocellulose, and Hybridization to Radioactive DNA DNA was digested with restriction endonucleases as specified by the suppliers and fractionated by electrophoresis in agarose (SeaKern)

Restriction Mapping of DNA Sequence Analysis Restriction maps of pSM4.3 and pS9E10.4 were constructed by elecirophoretic analysis of single and double digests of plasmid DNA. MtDNA fragment were cloned into M13mp vectors by shotgun or forced cloning techniques (Messing, 1983). DNA sequence analysis was by the dideoxy chain-termination method (Sanger et al., 1980) using [a32P]dCTP (410 CilmM; Amersham International). RNA Transcript Analysis and Sl Nuclease Mapping The procedure used for the fractionation of RNA was essentially that of Lehrach et al., (1977) as modified by Maniatis et al., (1982). MtRNA (5-20 pg) was electrophoresed under partially denaturing conditions in 1.3% (w/v) agarose and 17.3% (v/v) formaldehyde gels, transferred to nitrocellulose as described by Thomas (19809, and probed with 32Plabeled DNA (Messing, 1983). Sl nuclease transcript mapping was performed as described by Sharp et al., (19809, with the following modifications. Single-stranded Ml3 clones, complementary to the RNA transcript, were uniformly labeled by a modification of the sequencing reaction using a pentadecamer sequencing primer. The DNA was digested with an endonuclease to separate the cloned DNA from vector DNA, passed through a Sephadex G-50 spin column to remove unincorporated [32P]-dCTP (Maniatie et al., 19829, and the DNA was precipitated with 10 pg of E. coli tRNA and ethanol. The DNA was fractionated by electrophoresis in a 2% (w/v) alkaline low-melting temperature agarose gei that was wrapped in Saran Wrap and exposed to X-ray film for 30 min to 18 hr. The 3zP-labeled restriction fragment containing the insert DNA and a portion of the Ml3 vector was cut out of the gel and eluted (Maniatis et al., 1982). The 32P-labeled DNA (2500-20,000 cpm/assay), 5 pg mtRNA, and 10 bg E. coli tRNA were desiccated under

Cyrochrome 575

c Oxidase Subunit

I Genes

in Sorghum

vacuum and resuspended rn 2 PI of 2 M NaCI, 200 mM Pipes-NaOH (pW 6.4), and 5 mM EDTA by vortexing. The sample was heated at 85% for 5 min and submerged in a water bath at 45%. Hybridization was performed for 4-16 hr. Twenty-five units (250 U/ml) of St nuclease in 100 ui of ice-cold Sl nuclease buffer (250 mM NaCI; 30 mM NaOAC, pH 6.4; 1 mM ZnSO& and 20 pglml herring sperm DNA) was added, and the sample was incubated at 37% for 30 min. The .%-protected hybrids were precipitated with ethanol and analyzed on 8% (w/v) polyacrylamide-8.3 M urea DNA sequencing gels, in parallel with labeled molecular weight markers and an Ml3 sequencing ladder. Acknowledgments This work was supported by an S.E.R.C. Biotechnology Directorate grant and N.AT.0. Travel Grant to C. J. L., an N.I.H. Research Grant (GM29362) to T D. F., and an N.I.H. Fellowship to D. K. H. We are extremely grateful to 6. Dalton at Pioneer Hi-bred International for provision and maintenance of sorghum stocks, Dr. K. Schertz for obtaining seed with the genotype QE nucleus in QE cytoplasm, Dr. A. Tzagoloff for the yeast p- strain DS6/A422, and Dr. G. Schatz for antiserum prepared against yeast cytochrome c oxidase subunit I. We wish to thank Dr. R Isaac for advice, and A. Liddell, P Beattie, and X. Yong for their technical assistance. We thank Drs. D. Barker and A. Brennicke for confirming the sequence of the novel portion of the QE COXI ORF by Maxam-Gilbert sequence analysis. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received April 23, 1986; revised June 24, 1986 References Anderson, S.: Bankier, A. T, Barrell, B G., de Bruijn, M. H. L., Coulson, A. R., Drouin, J., Eperon, !. C., Nierlich, D. P., Roe, 8. A., Sanger, F., Schreier, R t-f., Smith, A. J. H., Staden, Ft., and Young, I. G. (1981). Sequence and organization of the human mitochondrial genome. Nature 290, 457-465. Anderson, S., de Bruijn, M. H. L., Coulson, A. R., Eperon, I. C., Sanger, F., and Young I. G. (1982). Complete sequence of Bovine mitochondriai DNA. J. Mol. Biol. 756, 683-717. Azzi, A. (1980). Cytochrome c oxidase. Towards a clarification of its structure, interactions and mechanism. Biochim. Biophys. Acta 594, 231-252. Batteiger, B., Newhall, W. J., V, and Jones, R. 8. (1982). The use of Tween-20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J. Immunol. Meth. 55, 297-307. Belliard, G., Vedel, F., and Pelletier, G. (1979). Mitochondrial recombination in cytoplasmic hybrids of Nicotiana by protoplast fusion. Nature 281, 401-403. Birnboim, H. C., and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513-1523. Boeshore, M., Liftshitz, I., Hanson, M., and Izhar, S. (1983). Novel composition of mitochondrial genomes in Petunia somatic hybrids derived from cytoplasmic mate sterile and fertile plants. Mol. Gen. Genet. 790, 459-467 Boeshore, M. L., Hanson, M. R., and Izhar, S. (1985). A variant mitochondrial DNA arrangement specific to Petunia stable sterile somatic hybrids. Plant Mol. Biol. 4, 125-132. Bolivar, F. (1978). Construction and characterization of new cloning vehicles ill. Derivatives of pBR322 carrying unique EcoRl sites for selection of EcoRI-generated recombinant molecules. Gene 4, 121-136. Bonen, L., Boer, F! H., and Gray, M. W. (1984). The wheat cytochrome oxidase subunit II gene has an intron insert and three radical amino acid changes relative to maize. EMBO J. 3, 2531-2536. Bonitz, S., Coruzzi, G., Thalenfeld, B., Tzagoloff, A., and Macino, G. (1980). Assembly of the mitochondrial membrane system: structure and nucleotide sequence of the gene coding for subunit I of yeast cytochrome oxidase. J. Biol. Chem. 255, 11927-11941.

Boutry, M., and Briquet, M. (1983). Mitochondr~al modifications associated with CMS in Faba Beans. Eur. J. Biochem. 727, 129-135. Burger, G., Striven, C., Machleidt, W., and Werner, S. (1982). Subunit I of cytochrome oxidase from Neurospora crassa: nucleotide sequence of the coding gene and partial amino acid sequence of the protein. EMBO J. 7, 1385-1391. Cabral, F., Solioz, M., Rudin, Y., Schatz, G., Clavilier, L., and Slonimski, P. P (1978). Identification of the structural gene for yeast cytochrome c oxidase subunit II on mitochondrial DNA. J. Biol. Chem. 253, 297-304. Chetrit, P, Mathieu, C., Muller, J. P., and Vedel, F. (1985). Mitochondrial DNA polymorphism induced by protoplast fusion in cruciferae. Theor. Appl. Genet. 69, 361-366. Davis, R. W., Simon, M., and Davidson, N. (1971). Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids. Meth. Enzymol. 27, 413-425. Dawson, A. J., Jones, V. P., and Leaver, 6. J. (1984). The apocytochrome b gene in maize mitochondria does not contain introns and is preceded by a potential ribosome binding site. EMBQ J. 3, 2107-2113. Dawson, A. J., Hodge, T. P., Isaac, P G., Leaver, 6. J., and Lonsdale, D. M. (1986). Location of the genes for cytochrome oxidase subunits I and II, apocytochrome b, a-subunit of the FrATPase and the ribosomal RNA genes on the mitochondriat genome of maize (Zea mays L.). Curr. Genet. 70, 561-564. de Bruijn, M. I-1. L. (1983). Drosophila melanogasfer mitochcndrial DNA: a novel organization and genetic code. Nature 304, 234-241. Dewey, R. E., Levings, C. S., Ill, and Timothy, D. H. (1985). Nucleotide sequence of the ATPase 6 gene of maize mitochondria. Plant Physiol. 79, 914-919. Dewey, R. E., Levings, C. S., 111,and Timothy, D. H. (1986). Novel recombinations in the maize mitochondrial genome produce a unique transcriptional unit in Texas male-sterile cytoplasm. Cell 44, 439-449. Dixon, L. K., and Leaver, C. J. (1982). Mitochondrial and CMS in Sorghum. Plant Mol. Biol. 1, 89-102. Duvick, D. N. (1965). Cytoplasmic 13, l-56. Edwardson, 341-420.

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Erion, J. L. (1985). Characterization of the mRNA transcripts of the maize, ribulose-1,5-bisphosphate carboxylase, large subunit gene. Plant Mol. Biol. 4, 169-179. Falconet, D., Lejeune, B., Quetier, F., and Gray, M. (1984). Evidence for homologous recombination between repeated sequences containing 18s and 55 ribosomal RNA genes in wheat mitochondrial DNA. EMBO J. 3, 297-302. Falconet, D., Delorme, S., Lejeune, B., Sevignac, M., Delcher, E., Bazetoux, S., and Quetier, F. (1985). Wheat mitochondrial 265 ribosomal RNA gene has no intron and is present in multiple copies arising from recombination. Curr. Genet. 9, 169-174. Forde, 8. G., Oliver, R. J., and Leaver, C. J. (1978). Variation in mitochondrial translation products associated with male sterile cytoplasms in maize. Proc. Natl. Acad. Sci. USA. 75, 3841-3845. Fox, T. D., and Leaver, C. J. (1981). The Zea mays mitochondrial gene coding cytochrome oxidase subunit II has an intervening sequence and does not contain TGA codons. Cell 26, 315-323. Galun, E., Arzee-Gonen, P., Fluhr, R., Edelman, M., and Aviv, D. (1982). Cytoplasmic hybridization in Nicotiana: mitochondrial DNA analysis in progenies resulting from fusion between protop!asts having different organelle constitution. Mol. Gen. Genet. 786, 50-56. Gergen, J. P, Stern, R. H., and Wensink, F? C. (1979). Filter replicas and permanent collections of recombinant DNA piasmids. Nucl. Acids. Res. i: 2115-2136. Isaac, P. G., Jones, V. P, and Leaver, 6. J. (1985). The maize cytochrome c oxidase subunit I gene: sequence, expression and rearrangement in cytoplasmic male sterile plants. EMBC J. 4, 1617-1623. Kyte, J., and Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105-132. Leaver, C. J., Hack, E., and Forde, B. G. (1983). Protein synthesis by isolated plant mitochondria. Meth. Enzymol. 97. 475-486.

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Leaver, C. J., Isaac, l? G., Bailey-Serres, J., Small, I. D., Hanson, D. K., and Fox, T D. (1985). Recombination events associated with the cytochrome c oxidase subunit I gene in fertile and cytoplasmic male sterile maize and sorghum. In Achievements and perspectives in mitochondrial biogenesis. vol. 11, E. Quagliarello, E. C. Slater, F. Palmieri, C. Saccone, and A. M. Kroon, eds. (New York: Elsevier), pp. 111-122.

Sharp, I? A., Berk, A. J., and Berget, S. M. (1980). Transcription maps of Adenovirus. Meth. Enzymol. 65, 750-768. Southern, E. (1975). Detection of specific sequences among DNA frag ments separated by gel electrophoresis. J. Mol. Viol. 98, 503-517. Thalenfeld, B. E., and Tzagoloff, A. (1980). Assembly of the mitochondrial membrane system: sequence of the oxi gene of yeast mitochondrial DNA. J. Biol. Chem. 258, 610-815.

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Thomas, P S. (1980). Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77, 5201-5205.

Levens, D., Ticho, B., Ackerman, E., and Rabinowitz, M. (1981). Transcriptional initiation and 5’termini of yeast mitochondrial RNA. J. Biol. Chem. 256, 5226-5232.

Wikstrom, M., Saraste, M., and Penttila, T (1985). Relationship between structure and function in cytochrome oxidase. In Tha Enzymes of Biological Membranes. vol. 4, A. N. Martonosi, ed. (New York: Plenum Press), pp. 111-148.

Levings, C. S., Ill, and Pring, D. R. (1976). Restriction endonuclease analysis of mitochondrial DNA from normal and Texas cytoplasm male sterile maize. Science 793, 158-160.

Note Added in Proof

Lonsdale, D. M., Hodge, T. P., and Fauron, C. M.-R. (1984). The physical map and organization of the mitochondrial genome from fertile cytoplasm of maize. Nucl. Acids Fies. 72, 9249-9261. Maniatis, T, Fritsch, E. F., and Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory). Manna, E., and Brennicke, A. (1988). Site specific circularization at an intragenic sequence in Oenothera mitochondria. Mol. Gen. Genet. 202, in press. Messing, J. (1983). New Ml3 Vectors for Cloning. Meth. Enzymol. 707, 20-89. Mikami, T, Sugiura, M., and Kinoshita, T (1984). Molecular heterogeneity in mitochondrial and chloroplast DNAs from normal and male sterile cytoplasms in sugar beets. Curr. Genet. 8, 319-322. Mueller, l? P, Reif, M. K., Zonghou, S., Sengstag, C., Mason, T L., and Fox, T D. (1984). A nuclear mutation that postranscriptionally blocks accumulation of a yeast mitochondrial gene product can be suppressed by mitochondrial gene rearrangement. J. Mol. Biol. 775, 431-452. Nagy, F., Torok, I., and Maliga, R (1981). Extensive rearrangements in the mitochondrial DNA in somatic hybrids of Nicotiana tabacum and Nicotiana knightiana. Mol. Gen. Genet. 783, 437-439. Nikolnikov, S., Posfai, G., and Sain, 8. (1984). The construction of a versatile plasmid vector that allows direct selection of fragments cloned into six unique sites in the cl gene of coliphage 434. Gene 39261-265. Norbrega, F. G., and Tzagoloff, A. (1980). Assembly of the mitochondrial membrane system. DNA sequence and organization of the cytochrome b gene in Saccharomyces cerevisiae D273-108. J. Biol. Chem. 255, 9829-9837. Palmer, J. D., and Shields, C. R. (1984). Tripartite structure of the Brassica campestris mitochondrial genome. Nature 307, 437-440. Pring, D. R., and Levings, C. S., Ill (1978). Heterogeneity cytoplasmic 127-136.

genomes

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male-sterile

cytoplasm.

of maize

Genetics

89,

Pring, D. FL, Conde, M. F., and Schertz, K. F. (1982). Organelle genome diversity in sorghum: male sterile cytoplasms. Crop. Sci. 22, 414-421. Rigby, R W., Dieckman, M., Rhoades, C., and Berg, F! (1977). Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I, J. Mol. Biol. 773, 237-251. Rothenberg, M., Boeshore, M. L., Hanson, M. FL, and Izhar, S. (1985). lntergenomic recombination of mitochondrial genomes in a somatic hybrid plant. Curr. Gent. 9, 615-618. Sanger, F, Nicklen, S., and Coulson, A. R. (1980). DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. Schardl, C. L., Lonsdale, D. M., Pring, 0. R., and Rose, K. R. (1984). Linearization of maize mitochondrial chromosomes by recombination with linear episomes. Nature 370, 292-298. Schardl, C. L., Pring, D. R., and Lonsdale, D. M. (1985). Mitochondrial DNA rearrangements associated with fertile revertants of S-type malesterile maize. Cell 43, 361-368.

The work referred to throughout as Bailey-Serres et al., submitted, is now in press: Bailey-Serres, J., Dixon, L. K., Liddell, A. D., and Leaver, C. J. (1986). Nuclear-mitochondrial interactions in cytoplasmic malesterile sorghum. Theor. Appl. Genet., in press.