SEQUENCE OF A MITOCHONDRIAL PLASMID OF SUNFLOWER (HELIANTHUS ANNUUS) AND ITS RELATIONSHIP TO OTHER MITOCHONDRIAL PLASMIDS
CHRISTINE PEREZ', BERNARD DUJON ~*, PHILIPPE HEIZMANN c and ANDRI~ BERVILLI~'**
°Laboratoire de G~n~tique, Station d'Am~lioration des Plantes, INRA, B Y 15/,0, ~103~ Dijon Cede:~ bContre de G~n~tique Mol~culaire, CNRS, 91190 Gif-su~ Yvette and "Universit~ Claude Bernard-Lyon I, Laboratoire de Biologic CeUulaire, Bonle~rd du II novembre 1918, 6962~ ViUeurbanne Cedez ?France)
(Received March 24th, 1988) (Revision received June 10th, 1988) (Accepted June 13th, 1988)
A circular plasmid called p i t is found in mitoehondria of sunflower (Helianthus an#uuz). Its nueleotide sequence has been determined and analyzed in order to understand its possible role and origin. The nucleotide sequence exhibits a 159 base pair region with highly organized repeats. The sequence of piT shows no major homology with other plasmids of higher plants although organized patterns ~u'e present in the circular plasmids. We also report the existence of two related mitocbendrial plasmids in a line of//. annuus and in the H. petiolarisfallaz species. In addition, piT plasmid has been detected in total cellular DNA of male-sterile sunflower with a copy number of a hundred times lower than in the male-fertile one.
The mitochondriai DNA of higher plants shows a typical organization which consists of a set of circular DNA molecules generated by recombinations from a master chromosome [1-4]. In addition to these high molecular weight DNA molecules mitochondria contain low molecular weight DNA ranging from 1 kb to 11 kb in size. They have been detected in many species such as in maize [5,6], sugarbeet [7], sorghum [8], broad bean [9,10], Brassica [3] and sunflower [11]. The largest of them are linear molecules in maize, Brassica and Sorghum ending in inverted repeats with a protein covently attached at their 5' extremi- ties. The other low molecular weight DNAs are smaller circular molecules that do not exceed 2 kb in size. They do not show significant homol-
*Present address: Institut Pasteur, Unit~ de g~n6tique mol~ulalre des levures, D~partement de biologie mol~cu- laire, 25 rue du docteur Roux, 75724 Paris C~lex 15, France. **To whom all correspondence should be addressed.
ogy with the main mitochondrial genome [12- 14]; consequently they are supposed to repli- cate autonomously and so are called mitochondrial plasmids. No evident features common to all these minicircles have been found and their relation, if any, with the main high molecular weight genome remains unclear. The origin and the role of these circu- lar plasmids have not yet been elucidated. Evidence for transcription has been reported in sugarbeet, faba bean and maize [13,15-18].
In Helianthus annuus, a circular 1.45 kb DNA molecule has been described in mitechon- dria [11,19]. This plasmid molecule is observed with maie-fertfle cytoplasm but cannot be detected by agarose gel electrophoresis in nuclear isogenic lines with Leclercq male ste- rile cytoplasm [20~21]. This difference makes the plasmid a potentially useful marker of the two cytoplasms. We were therefore interested in the molecular characterization of this plas- mid. We report here the sequence of the plas- mid and its analysis.
Plant material Seeds of Helianthus annuus fertile lines
HA89 and CANP3 were provided by Agri-obten- tion SA. These two lines carried H. annuus cytoplasm and had nuclear maintainer genes for the cytoplasmic male sterility (CMS) Leclercq [22]. The two ecotypes 200 and 674 of the fallax subspecies of Helianthus petiolaris were provided by Serieys (INRA, Domaine de Melguefl, Mauguio 34130, France).
CMS occurs when H. annuus nuclear back- ground is introduced into cytoplasm from the related species H. petiolaris [22,23]. Lines with nuclear background that maintains the "Le- clercq" CMS are called A type if they are on the "Leclercq" CMS cytoplasm (they are male-ste- rile) or B type if they are on H. annuus cytoplasm (they are male-fertile).
Preparation of mitochondrial and chloroplast DATA
We worked out a technique for obtaining mitochondrial DNA preparations enriched in mtDNA plasmid molecules. The method we used to isolate mtDNA from etiolated young plants or from leaves has been adapted from the method used for chloroplast isolation [24] and from Davis [25].
Etiolated plants were obtained after grow- ing them ten days in the dark. Mature plants are grown in a greenhouse. The following data are given for approx. 100 g of leaves.
Material is ground in a blendor with 400 ml of the following extraction buffer, 0.05 M Tris -HC1 pH 8.0, 0.025M Ethylene-diaminete- traacetic acid (EDTA), 1.3 M NaCl, 0.056 M 2- mercaptoethanol and 2°/0 half skimmed milk. Then the filtered liquid is centrifuged 15 rain at 4500 rev./min; the pellet is used to extract the cpDNA if necessary, the supernatant is then centrifuged 15 rain at 12 000 rev./min to sedi- ment mitochondria, which are then treated with 1.7 mg of DNAse I in 20 ml of buffer, 0.05M Tris--HCI pH 8.0, 1.3 M NaC1, 0.005 M magne- sium acetate for 1 h at 0 °C. The DNase reaction is stopped with the addition of 70 to 100 ml of
the extraction buffer. Mitochondria are pelleted by a 15 min centrifugation at 12 000 rev./min. Pellets of chloroplast or mitochondria are then lysed in 15- 20 ml lysis buffer (0.025 M Tris-HCl pH 8.0, 0.012 M EDTA, 30/0 SDS, 0.15 M 2-mercaptoethanol, and 500 ~g/ml diethylpyrocarbonate (DEPC)) incubated 20 min at 37 °C and then cooled at 0 °C. Protein- SDS complexes are precipitated at 0 °C for 20 min with 1.2 M potassium acetate and dis- carded as a pellet after a 10 min centrifugation at 10 000 rev./min. The supernatant is extracted once with phenol-chloroform and twice with chloroform and then precipitated in 0.5 M ammoniun acetate with absolute ethanol and precipitated once more in 0.3 M sodium acetate with absolute ethanol. Nucleic acids are treated with a mix of RNAse A and RNAse T1 followed by a chloroform step and an ethanol pre- cipitation.
We can visualize plasmid DNA from the analysis of mtDNA corresponding to 3 - 5 g wet weight of leaves.
Preparation of total cellular DNA Total DNA has been prepared from approx.
50 lyophilized etiolated germs of CANP3 B and CANP3 A lines of sunflower as described [26].
Cloning of the plT plasmid The 1.45-kb mitochondrial plasmid DNA
from CANP3 B line (called plT) was purified from the high molecular weight mitochondrial DNA by a 10%-30% sucrose gradient in 0.03 M Tris--C1, 0.01 M EDTA, pH 7.6. Fractions were analyzed by agarose gel electrophoresis and those which contained the small circular DNA were pooled. The p i t plasmid DNA was restricted by EcoRI restriction endonuclease and cloned in the EcoRI site of pEMBL8( - ) in E. coli strain 71/18. Transformation was per- formed by the CaCI2 method as described [27]. Amplification of the recombinant plTC20 plas- mid was carried out in 100 ml of LB culture with ampicillin (50 ~g/ml). Large scale prepara- tions were performed either by the alkaline method or by the "boiling method" [27].
Sequencing of the plasmid The sequence was determined by the Maxam
and Gilbert sequencing method [28].
Probes (i) piT insert was purified from pEMBL8( - )
vector by agarose gel electrophoresis and nick- translated with [o-nP]ATP [27]. (fi) The 1,2-kb PstI-EcoRI fragment of piT plasmid was cloned in M13 tg 130 phage and used to prepare a-s2P singie-stranded probes [29].
Hybridization DNA fragments were transferred to nitro-
cellulose filter [27] and hybridized in 3 x SSC (1 x : NaC1 0.15 M, sodium citrate 0.015 M, pH 7.0), 5 x Denhardt's solution (1 x : 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.020/0 bovin serum albumin), 0.1% sodium dodecyl sulfate, 200 ~g/ml sonicated denatured salmon sperm DNA and o- ~Plabelled probe. Hybridization was performed at 65°C without formamide or at 42 °C with 500/0 formamide for 16 h. Washing was done in 0.5 x SSC, 0.1% SDS at 63°C (Fig. 3) and 1 x SSC, 0.1% SDS at 63°C (Figs. 5 and 6).
Results
Characterization of plasmids present in prepa- rations of mitochondrial DNA from different lines of species
Agarose gel electrophoresis of DNA allowed us to observe a single band with apparent mobility of approx. 1 kb (Fig. 1). The band was purified from the mt DNA through a sucrose gradient, 10%--30% linear (w/v). The super- coiled circular form of a 1450 bp contour length plasmid was characterized by electron micrc~ scopy (Fig. 2A) and by gel electrophoresis.
Figure 3 gives examples of DNA prepara- tions of different ecotypes chosen under the fol- lowing criteria: the "Leclercq" CMS widely used for sunflower hybrid production was origi- nally obtained from an interspeeific cross between Helia#thus annuus and a Helianthus petiolaris, a closely related species used as female parent [22~ Since the original H. pet/o/ar/s form is no longer available we
61
1 2
m
2.0 1.6 ¢ 1.4 0 . 9
Fig. 1. Unrestricted mitochondrial DNA of H. annuus
hypocotyls. Sample was electrophoresed on an 1% agarose gel. Lane 1: DNA molecular weight marker; ;t DNA restricted with HindIII and EcoRI. Lane 2: Mitoehondrial DNA preparation from HA89 B line; (*-) chromosomal mt- DNA; ( • CCC piT plasmid form.
examined two ecotypes of Helianthus petiolaris ssp. falla~ the ecotypes 200 and 674, instead, an interspecific cross between H. annuus and H. petiolaris fallaz 200 has given rise to a new CMS which is called "fallax" CMS [23].
Bands with apparent mobilities correspond- ing to 1.0 kb and 1.6 kb are detected in lines HA89 B and CANP3 B on agarose gels (Fig. 3A). When piT plasmid from CANP3B is used as a probe (Fig. 3B), the two bands hybridize indicat- ing that they are two forms of the same plasmid. The 1.0~kb form has already been described and represents the supercofled form of a 1.4~kb plasmid. The 1.6-kb form which is observed in some preparations but not all (see Fig. 1) probably represents the open circular form of the same plasmid. Both lines HA89B and CANP3B contain the same plasmid.
In addition, CANP3 B mitochondria contain two other less abundant bands with mobflities of 1.2 and 1.8 kb that also hybridize with the plTC20 probe and lead us to consider the pres- ence of another low molecular weight DNA (plC) with a sequence related to piT. The plC plasmid is not visible in HA89B preparation.
The two ecotypes 200 and 674 of H. petiolaris
62
® • o . 2 ~
®
Fig. 2. Electron microscopy of piT and plF plasmids. (A) Preparation of HA89 B mitochondrial plasmids purified from mitochondrial DNA by a sucrose gradient. (B) Is a preparation of the band with mobility of 2.6 kb of H. petiolaris fallax 200 mitochondrial DNA electroeluted from an agarose gel.
® ® 1 2 3 4* 4 5 6 7 1 2 3 4* 4 5 6 7
2.3D, 2.0D,
1.6 IP 1 .3D
0.98~ 0.83b
9 '
ssp.falla~ have three plasmid DNA species with mobilities of, respectively, 0.8 kb, 1.4 kb and 2.6 kb (Fig. 3A). The two larger cross-hybridize with the plTC20 probe, the 0.8 kb band does not. Fig. 2B shows that the molecules of the 2.6 kb band are in the open circular form. The esti- mated size of that plasmid (called plF) is 2150 bp as measured on electron microscopy pic- tures. The 1.4 kb probably corresponds to the supercoiled form of that plasmid.
No cross-hybridization with chloroplast DNA (lane 6 Fig. 3) of H. petiolazis fallaz ~00 is observed.
Sequencing o/piT The EcoRLEcoRI insert of 1.45 kb in clone
plTC20 has been sequenced by Maxam and Gil- bert method. The nucleotide sequence contains 1415 bp (Fig. 4), with a G-C content of 45.4%, not significantly different from that of mitoehondrial DNA at 46.9% [11]. This is simi- lar to other plasmids of higher plants [15,18] except for the linear plasmids S1 and $2 of maize, which contain 37.5% G-C [30,31].
Nucleotide sequence analysis Part of the plasmid sequence (244--403) con-
tains multiple tandem repeats that can be sub- divided in units (Fig. 4). These blocks always occur in the same order, but are organized in higher order repeats (A,B,C~D,I, see Fig. 7). They are often separated by single nucleotides that differ between the high order repeats. Two blocks are also found elsewhere in the sequence as a direct repeat (al block 745-752) or inverted (a2 block 8 2 5 - 833).
An A-T rich region forms an inverted repeat
6 3
l GAATIC]F~ GI'~CCCTC~A
51 AAAAGACAAA G~AAGGCGGT
]01 TF~CTFTTTA AICfCAAAAG
151 1GCAGCTA1C ATIATC6GGG
201 CCfCG~CTGC AG'iCGAGGGT a2 b l
251 .~TTTTACT ~TACAGAA al a2 T
301 CA~TCGACC 41TTTTTAC I I ~ '4 "~
351 AGAGA~IA6 CAqATCGACC
401 ~AAAAAG 6TTCNAATGA
451 GAGCTTTGTA ~AGGAGTAGA GAGTGTAAGA
501 GCGAGTTGGG TAG~CACCTC TCCAACCCAT
~51 GCCCCCTAGC AGCGCTCTCG AAACTCTACA
bOl CGATTGAGTT AACGGAAGC~ TCrCAAAAAC
~51 AGAAGAAGAC TCAGCGCTGG GTAAGTGGTT
701 CTCGGC]CCT TGGGGAGTAG TGCCCCCAAG
751 C~CGCGGCT CCATACCCCT AG~TTAG]AT
801 TTTTTCT~CTG AGGAGCAAAG AAA~GT~A
851 GGACG6CCAG CCCGGTAGGA AG6TGAGATA
901 ~ ' ~ 3 ~ T C TGTFAAGGqC GAGTCCFIAC
951 C~6ACTCGGC ")'GGACTTGTT AGCTGCATAA
1"01 CGGAGAATGA AGATCACCCG AACITCATTC
1051 AGCAG~AGAA GCTCTTCTCT TTGCTGGCCA
1101 CCCCCCCCCT CCCATGGC~T GCTTFGTT~C
t lS l I'AGCTGACAA 6TCCGCTCTC CCCGACTIAA
]201 AACAGCTCTA TATACTAGGG GA~GAGAGCG
1251 ~CAAC~AGAG CATIG~TTG TG~TCCGGGi"
1301 ~ACTCCTTqC TT~TTGA~6~ T'['iACGGCCT
1351 GICICAAA~G G[ACCCI'A'I'A ITSAICTTGA
i~i ~TCAAGA]GG ICNT~
CA~6C~AC~A AIC.C~AGG~ AAGAAAA!~
TTTGAGACAT AAAAAATAAA TGCTAAA[
GTCGATCTI~ TTGTTT~GHG ~C~CAA~
TGC TCCGCCA CCCC TCGT r T CCA TC* f ~
A CG I CTCC LIT TCCCT AA~AA A~ a~'~'~ I I b~ I I b~ /
Pig. 4. The sequence of the piT plasmid obtained from clone plTC20. The sequence of the plasmid has been linear- ized at the EcoRI site wh/eh has not been sequenced across. The 159 bp repeat region and the two repeated patterns al and a2 are boxed. The solid underline identifies the A - T rich region and broken underlines indicate the two G - C rich regions. Arrows show inverted repeated sequences.
Fig. 3. Comparison of mitochondrial DNAs from H. annuus and H. petiola6.& (A) unrestricted mitochondrial and chloroplast DNA ofH. annuus and H. petiolarisfallaz electrophoresed on an 1% agarose gel. Lane 1: a mix of A DNA digested by HindIII and I DNA digested by HindIII and EcoRI. Lane 7: BRL I kb ladder DNA. Lanes 2 - 5: mitochondrial DNA from hypocotyls of H. annus HA89 B line (2), CANP3 B line (3) and from leaves ofH. petiolazisfallaz 200 (4 and 4"}, 674 (5). The mtDNA in lane 4* has been prepared as described in Materials and methods except that contrifugation after SDS-potassium acetate precipita- tion was at 4500 rev./min instead of 10 000 rev./min. Lane 6 contains chloroplast DNA from H. petiolarisfallo.z 200. B: Southern transfer of gel A probed with a 32P nicktranslated plTC20 clone insert. The higher MW hybridizing bands in lanes 3, 4*, 4 and 5 are unspecific signals due to the large amounts of mtDNA (5-- 10 ~g) loaded on the agarose gel in order to visualize the piT, plC and plF plasmids through EtBr fluorescence. Lane 7: the 1.6 kb fragment signal of the I kb ladder is due to the homology with the Pembl 8 vector according to the manufacturer. Symbols show the bands of mobility of piT ( • ), of plC ( <J ) and of H. petiolarisfallaz plasmids ( • ).
64
( ~ EcoRI Bgll BstEII Pstl / ~ EcoRI Bgll BstEII Pstl C M A B A B A B A B M C M A B A B A B A B
~ii!iiiiii
| g"
Fig. 5. Detection of pi t sequences in total DNA from CANP3A (A) and CANP3B (B) digested with four restriction endonu- cleases. (A) EtBr stain agarose gel electrophoresis of DNA. Lane C: clone plTC20 cleaved by EcoRI. M: ~ DNA digested by BstEII. (B) Southern transfer of gel shown in A, hybridization with the large EcoRI-PstI pit fragment used as a probe. Arrows indicate the three forms of piT: linear (b), open circular (a), supercoiled (c) and a potentially multimeric form (d).
f rom 817 to 849 (940/0 A - - T ) . Two G - C rich reg ions a re p r e s e n t wi th 90.50/0 G - C ( 1 0 9 3 - 1113) and 90.9°/0 G - C ( 8 8 6 - 908).
No la rge ORF can be found if the t h r e e s top codons of the un iversa l code are considered. The l a rges t r e a d i n g f r a m e is only 372 bp long and s t a r t s wi th an A T G codon. The second larg-
es t r ead ing f r a m e is 333 bp long but does not have an ATG codon.
Detection of p lT plasmid in total cellular DNA of CANP3B and CANP3A lines
The p i t p lasmid has been o b s e r v e d in D N A p r e p a r e d f rom mi tochondr ia of male fer t i le B
kb
4 . 3 2 .
3.6S.
2 .32.
! . 9 3 .
1.37.
1.26--
M 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 a
U
65
Fig. 6. Detection of piT plasmid in total cellular DNA from CANP3 B and CANP3 A lines. (A) Agarose EtBr stain gel electro- phoresis of total DNA preparations from the two lines. Lane 1: undigested CANP3 B DNA. Lane 2 - 8 : EcoRI digested DNA. Lane 2:CANP3 B DNA. Lanes 3 - 6 are mixtures of CANP3 B DNA and CANP3 A DNA preparations respectively (CANP3 B/ CANP3 A), 10 -1 (lane 3), 10 -8 (lane 4), 10 -8 (lane 5) and 10-' (lane 6). Lane 7: a 10 -5 dilution of CANP3 B in water. Lane 8: DNA of CANP3 A. Each sample (1-- 6 and 8) contains 10 ~g of DNA. Arrows show the different forms of piT molecule: the open circular (a), the linear (b) and the supercoiled formes (c). A band (d) corresponds to a multimeric form of p i t since it disappears with digestion. B: Southern transfer of gel A probed with the large PstI-EcoRI piT fragment (the film has been exposed 25 h with intensifying screen at - 70 °C).
lines but could not be detected previously in male sterile A lines by ethidium bromide fluc~ rescence [11,19,21]. To clarify this question we prepared total cellular DNA from both A and B lines and probed them with plasmid piT (Figs. 5 and 6). Undigested DNA of line B or DNA cleaved with enzymes that do not cut piT, shows the three forms of the plasmid as expected
(a,b,c). A fourth minor band (d) may correspond to a multimer since it disappears after digestion with enzymes that cleave piT, EcoRI or PstI. The DNA of line A also shows an hybri- dization signal at the position of the linearized plasmid (b) (Fig. 6) or at the position of the open circular form (a) (Fig. 5) although with much lower intensity than that of line B. To examine
66
@ 244
C A O C
1226
T TCC
C
®
bl
403
T T A T C T T A G C
D A B I
al = A T C G A C C G a2= T T T T T A C TT
bl = T T A C A G A A G A G A G A b2= A G C A C
10b
1373
G G C T G T T T C T ACA C G T T C O T
B, A B2 D C
E
al = A A T T
a2 = T T A A A T T A A T T
bl = A A A A G A
b2 = C A A G A A T A
lOb
1501 ~ 1 32
T A A T A AG T A AT A T A AGA A
m~lblclldll a2 I lbl¢2] al llblcll[ i--Jibl=, ] -,1 al Ilblclld!l aZ
B D A C
al = ACTTGCTCCTCCGTCGC lob
a2 = GCTCCTACGTCGC
b = AGCT
cl = TA~AATC c2 = AAAAAGCG d --. TTCCT
Fig. 7. The organization of tandemly repeated regions in sunflower piT plasmid (1) compared with 2 possible constructions proposed for the sugarbeet Mc. a plasmid (2) and plasmid I of faba bean (3).
the relative copy number of plasmid in lines A and B we mixed DNA in various proportions (Fig. 6). Hybridization signals become compara- ble to that of line A in the 1/100 dilution, indi- cating that the number of plasmid molecules per cell is roughly 100 times higher in line B than in line A.
Discussion
We report here the sequence of a circular
plasmid (piT) isolated from mitochondria of H. a n n u u s CANP3 B. The sequence has been determined from a recombinant clone contain- ing a 1415 bp long EcoRI fragment believed to represent the entire pi t plasmid since the size of the fragment is in good agreement with the size of open circular molecules measured by electron microscopy.
The sequence of piT contains numerous stop codons in all six potential open reading frames. The largest protein product predicted would
67
contain only 124 amino-acids from the first in frame AUG codon. The absence of large ORFs is a common trait of all small circular mito- chondrial plasmids of higher plants sequenced so far. The longest possible ORFs in the 1.94 kb maize plasmid are only 81 codons [13], and 112 codons in the sugarbeet Mc. a plasmid [17]. The linear plasmids of maize on the other hand contain long ORFs and the translation product of one of them has recently been identified [16].
A 159 nucleotide long region is organized with many duplications and short patterns; we found similar organizations for other circular plasmids (Fig. 7) in the 1620 bp Mc a of sugar- beet the region is 142 nucleotides, in the 1704 bp Mc I of field bean the region is 234 bp long, [15,17,18].
The sequence of piT has been compared with the higher plant mitochondrial plasmid se- quences available: sugarbeet plasmids Mc.a, Mc.c and Mc.d [15,17], Vicia faba plasmids 1, 2 and 3 [18], maize plasmids 1.94 kb [13], $1 and $2 [30,31] without revealing extensive homology with any of them. The most significant se- quence homology found is a short sequence (57 bp) with homology with Vicia faba plasmid 1 (Fig. 8). It extends from position 713 to 768 of the p i t sequence and shows 81.5O/o homology with the Viciafaba.
Parts of the B pattern sequence of piT show homology with a 21 nucleotide sequence of Mc.c (Fig. 7C) sugarbeet, an 18 nucleotide sequence of faba bean plasmid 2 and a 16 nucleotide sequence of faba bean plasmid 3 (respectively, 76%, 77.50/0 and 72.20/0). The two sequences of faba bean are contained in the 47 nucleotide region of 94% homology between them.
Computer screening testing in the piT sequence against sequence data from "EMBL" and "Gene Bank" libraries failed to reveal any significant homology.
768 GGGTATG~A~SCC,GCGTCG~FCGA~GGC~TGGCTGTGTCTTGGGGC~T,A~TCCC 71~ ~Itflllillll illl!l if| I illf lli! llill III III~I
Fig. 8. Region of homology between nt 713--768 of the sunflower plasmid piT and nt 1264-- 1318 of plasmid 3 from faba bean mitochondria.
The plasmid piT can be found in different B lines of H. annuus, two of which (HA89 and CANP3) have been studied here. In one of them, CANP3, a second plasmid plC differing from piT in size could also be detected. Cross hybridization between piT and plC indicating common sequences suggests that they either derive from a common origin or one of them is derived from the other. The piT sequence could be included entirely in plC. A similar situation has been reported in maize mitochondria where in some isolates a 2.1 kb plasmid is a deleted form of a 2.3 kb plasmid found in other lines [32]. The same is also true in Vicia faba with plasmids 1540 and 1700S [33].
In H. petiolaris faUaz another plasmid (plF) is present in two forms in the mitochondria of the two ecotypes studied here. This plasmid also cross hybridizes with piT indicating com- mon sequences. This homology is in agreement with the idea that H. petiolaris is one of the most closely related species of H. annuus [34]. Other faint bands can also be observed by ethi- dium bromide fluorescence in mitochondrial DNA preparations, of ecotypes 674 and 200 which have not been characterized further but may represent a population of plasmid mole- cules heterogeneous in size and sequence within the H. petiolaris fallaz and H. annuus species.
In maize [12], sugarbeet [17] or sorghum [35] no copy of the small circular plasmid could be found integrated in the main mitochondrial genome. Our hybridization experiments (Fig. 3) using piT probe also give no positive evidence for the presence of integrated copies of piT. The low non-specific hybridization of the probe possibly could mask the presence of low copy number sequences related to piT, but the result clearly rules out the possibility of numerous integrated copies of piT.
A strong hybridization signal is found at the appropriate molecular weight as expected, when total cellular DNA of lines CANP3 B of H. annuus is probed with piT. The same hybridiza- tion pattern is found in line CANP3 A, except that the intensity of the signal is approx. 100 times lower. This is in contradiction to the pre- vious idea that the p i t plasmid is absent from
68
A lines. This result can be interpreted in two ways: either each plant of the A line contains the piT plasmid in low copy number of the population of A type seeds used to prepare DNA was heterogeneous with contaminant B type seeds present. The preparation of total DNA from individual seeds is necessary to solve the problem in the future. The sensitivity of detection of p i t probe on total cellular DNA moreover shows this tes t to be useful to characterize batches of A or B lines of sun- flower.
Acknowledgements
We wish to thank C. Schneider for his help in electon microscopy and J. Delbut for photo- graphic prints. This work has been supported by a grant 861007 from INSERM to B. Dujon and by a grant 854439 from INRA and MRT to A. Bervill~.
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