Volume 14 Number 14 1986
Nucleic Acids Research
Tripartite mitochondrial genome of spinach: physical structure, mitochondria] gene mapping, and
locations of transposed chloroplast DNA sequences
David B.Stern1* and Jeffrey D.Palmer2
Carnegie Institution of Washington, Department of Plant Biology, 290 Panama Street, Stanford,
CA 94305, 'Department of Botany, University of California, Berkeley, CA 94720 and 'Department of
Biology, University of Michigan, Ann Arbor, MI 48109, USA
Received 19 May 1986; Accepted 20 June 1986
ABSTRACT
A complete p h y s i c a l map of t h e spinach m i t o c h o n d r i a l genome has been
established.
The entire sequence content of 327 kllobase pairs (kb) is
postulated to occur as a single circular molecule. Two directly repeated
elements of approximately 6 kb, located on this "master chromosome", are
proposed to participate in an intragenomlc recombination event that
reversibly generates two "subgenomic" circles of 93 kb and 234 kb. The
positions of protein and ribosomal RNA-encodlng genes, determined by
heterologous f i l t e r hybridizations, are scattered throughout the genome,
with duplicate 26S rRNA genes located partially or entirely within the 6 kb
repeat elements. Filter hybridizations between spinach mitochondrial DNA
and cloned segments of spinach chloroplast DNA reveal at least twelve
dispersed regions of interorganellar sequence homology.
INTRODUCTION
M i t o c h o n d r i a c o n t a i n t h e i r own DNA, w h i c h e n c o d e s p a r t s of t h e
o r g a n e l l a r t r a n s l a t i o n a l a p p a r a t u s and components of t h e e l e c t r o n t r a n s p o r t
chain.
Genes e n c o d i n g 26S, 18S and 5S rRNA ( 1 - 3 ) , s u b u n l t s I and I I of
c y t o c h r o m e o x i d a s e ( 4 , 5 ) , s u b u n i t s 6 ( 6 ) , 9 (7) and a l p h a (8) of t h e
n i t o c h o n d r l a l ATPase, a p o c y t o c h r o m e B (9) and a gene homologous t o URF-1
(10), an u n i d e n t i f i e d reading frame found i n animal (11) and fungal (12-14)
mitochondria, and s e v e r a l tRNAs (15, 16) have been I d e n t i f i e d and sequenced
for one or more plant mitochondrial genomes (for reviews of plant
mitochondrial genes and genome s t r u c t u r e , see r e f s . 17 and 18). Plant
mitochondrial DNA (mtDNA) also contains substantial regions of homology to
chloroplast DNA (ctDNA; 19-21). No functions have as yet been ascribed to
t h i s "promiscuous" (22) DNA. Plant mtDNAs are distinguished from t h e i r
fungal and animal counterparts by their large size (23) and by the presence
of repeated DNA elements, certain of which appear to be Involved in interand intramolecular recombination events (24-27).
In turnip mtDNA,
Intramolecular recombination is postulated to generate a t r i p a r t i t e genome
s t r u c t u r e (27), whereas the corn m i t o c h o n d r i a l genome e x h i b i t s a
multicircular organization that is proposed to result from recomblnational
activity at five major pairs of repeated elements (26).
In the present work, we describe the organization of the 327 kb spinach
mitochondrial genome. I t s proposed t r i p a r t i t e structure is very similar to
© I RL Press Limited, Oxford, England.
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that
of
turnip
mtDNA (22).
both bona fide
scattered
Heterologous
mitochondrial
throughout
filter
hybridizations
reveal
that
genes and regions homologous with ctDNA are
the genome.
MATERIALS AND METHODS
Methods used for the p u r i f i c a t i o n ,
gel
electrophoresis
and f i l t e r
mtDNA have b e e n d e s c r i b e d
prepared by a modified
DNA c l o n e s
Sal
I,
of spinach
( 2 0 , 28,
alkaline
29).
lysis
endonuclease
Plasmid
and c o s m i d
(30) procedure.
f o l l o w e d by l i g a t l o n
(31) and t r a n s f o r m a t i o n
digestion,
of spinach (.Spinacla
oleracea)
DNAs were
Recombinant
mtDNA were generated by d i g e s t i o n
P s t I or Kpn I ,
plasmid vector
restriction
hybridization
plasmid
of the mtDNA with
t o an a p p r o p r i a t e l y
(32) i n t o E. coll
c u t pUC
s t r a i n JM83.
A
s p i n a c h mtDNA c o s m i d l i b r a r y , e s t a b l i s h e d i n pHC79 ( 3 3 ) , was s c r e e n e d and
maintained
as described (34).
S p i n a c h ctDNA c l o n e s
used
i n h e t e r o l o g o u s f i l t e r h y b r i d i z a t i o n s were
d e s c r i b e d i n an e a r l i e r p u b l i c a t i o n ( 3 5 ) .
M i t o c h o n d r i a l gene p r o b e s f o r
s u b u n i t s I and I I o f cytochrome c o x i d a s e , t h e 26S, 18S and 5S rRNAs, t h e a
subunit and subunit 6 of the mitochondrial ATPase, apocytochrome B and URF-1
are d e s c r i b e d i n d e t a i l
in the t e x t and i n Table 1.
RESULTS
P h y s i c a l Map of Spinach mtDNA
In order
genome,
to establish
a restriction
map of the spinach
mitochondrial
p l a s m i d DNA c l o n e s were randomly s e l e c t e d from mtDNA l i b r a r i e s
generated w i t h Sal I, Pst I and Kpn I.
S m a l l - s c a l e plasmid DNA preparations
were analysed by r e s t r i c t i o n endonuclease
d i g e s t i o n and g e l e l e c t r o p h o r e s i s
to e s t a b l i s h a s e t of unique clones for each enzyme.
These c l o n e s were
32
subsequently l a b e l l e d with
P and hybridized to f i l t e r b l o t s containing
single and double digests of spinach mtDNA.
An example of this mapping strategy i s shown in Fig. 1.
cloned
Sal
fragments
I fragment of
of
hybridizes
6.45 kb i s
IB, lane SK).
by Pst I (Fig.
P-labelled
into
The clone, pJDP20,
with Kpn I fragments of 22 kb and 18.1 kb (Fig.
linkage.
32
shown to be cleaved by Kpn I
3.35 kb and 3.10 kb (Fig.
demonstrating their physical
fragment of
A
IB,
lane K),
The Sal I insert of pJDP20 i s uncut
IB, lane SP), and the clone hybridizes with a s i n g l e Pst I
13.5 kb (Fig. IB, lane P).
Thus, t h i s 6.45 kb Sal I fragment
overlaps both the 22 kb and 18.1 kb Kpn I fragments, but i s i n t e r n a l to a
13.5 kb Pst I fragment.
The physical map was extended by similar
analyses
of plasmid clones containing a total of 67% of the genome.
Since the restriction map produced by f i l t e r hybridizations of plasmid
clones was incomplete, as judged by the number of Sal I fragments v i s i b l e in
an ethidium bromide-stained g e l (Fig.
1A, lane S) but not present on the
preliminary linkage map, a cosraid DNA library consisting of 384 clones with
35-45 kb i n s e r t s
of
partial
Sau3A d i g e s t i o n products vas
constructed.
Cosmlds were s e l e c t e d from the l i b r a r y by t h e i r h y b r i d i z a t i o n with
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Nucleic Acids Research
SK K P SPS SKK
122
181
13-5
645
335
310
Figure 1. Mapping s t r a t e g y for plasmid clones of spinach mtDNA. Spinach
mtDNA was digested with Pst I (P), Sal I + Pst I (SP), Sal I (S), Sal I +
Kpn I (SK) and Kpn I (K), electrophoresed in a 0.7% agarose gel, stained
with ethidium bromide (left panel) and transferred to a GeneScreen f i l t e r .
The f i l t e r was hybridized with the n i c k - t r a n s l a t e d clone pJDP20, which
c o n s i s t s of pUC8 and a 6.45 kb Sal I fragment of spinach mtDNA (right
panel). Size markers are in kb, and were determined from Sal I, Eco RI and
Hind I I I digests of phage lambda (A) DNA.
l a b e l l e d r e s t r i c t i o n fragments from the ends of previously characterized
linkage groups.
Each cosmid was placed on the r e s t r i c t i o n map, and
simultaneously verified for integrity by the strategy i l l u s t r a t e d in Fig. 2.
The cosmid clone cSIIElO was digested with Sal I, Pst I or Kpn I,
electrophoresed in an agarose gel next to a lane of spinach mtDNA digested
with the same enzyme, t r a n s f e r r e d to n i t r o c e l l u l o s e , and probed with the
in
P-labelled cosmid. Since the cosmid vector has one Sal I site and one Pst
I site (33), each of these enzymes generates two vector-containing fragments
which do not align with genomlc DNA fragments (Fig. 2, lanes 1 and 3, marked
V).
Kpn I does not cleave the vector, so only a single vector-containing
fragment is expected (Fig. 2, lane 5). The remaining DNA fragments, which
are fully contained within the cosmid insert, both hybridize and align with
the corresponding fragments in the mtDNA digest (e.g. 12.5 kb, 8.1 kb and
2.3 kb Kpn I fragments; Fig. 2, lanes 5 and 6). The cosmid also hybridizes
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Nucleic Acids Research
S
P
cos rrt
K
cos mt cos mt
36
2-3
Sal II
PstI
Kpnl
36
&5 |
&8
Nil I 68
I,|23|IJ81
|
I „
INI
125
222
|42 | &5 |
I 114
I
IIE10 • -
Figure 2. Happing strategy for cosmid clones of spinach mtDNA. 0.2 pg of
cosmid csIIElO DNA (lanes marked "cos") and 1.5 /ig of spinach mtDNA (lanes
narked "rat") were digested with Sal I (S), Pst I (P) or Kpn I (K),
electrophoresed in a 0.7% agarose gel, transferred to GeneScreen and
hybridized with nick-translated csIIElO DNA. Cosmid DNA fragments
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Nucleic Acids Research
consisting partly of vector sequences are indicated with a "v". Spinach
fragments which are partially included within the cosmid insert ("border"
fragments) are marked with a "b". The bottom of the figure shows a
restriction map of the spinach mtDNA region cloned in csIIElO, and below it,
the extent of cosmid csIIElO. Sizes of fragments are in kb, and were
determined as in Fig. 1.
with two genoraic DNA fragments not fully contained within its insert (e.g.
36 kb and 22 kb Sal I fragments; Fig. 2, lane 2, also lanes 4 and 6, marked
"b"). The combined results of hybridizations with overlapping cosmid and
plasmld clones led to construction of short restriction maps such as that
shown at the bottom of Fig. 2, which could be linked together. Eventually,
cosmid and plasmid clones covering over 96% of the genome were analysed, and
the physical map was ascertained to be complete.
A restriction map of the spinach mitochondrial genome is shown in Fig.
3. The genome exists as a circular molecule approximately 327 kb in length.
The map accounts for every Sal 1 and Kpn I fragment visible in a stained gel
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Figure 3. Restriction map of the spinach mitochondrial genome. Fragment
sizes are in kb. A zero point of linearization was arbitrarily selected as
the site separating the 14.5 kb and 1.7 kb Sal I fragments. The relative
order of certain clustered Kpn I and Pst I fragments has not been
determined. Two doublet Pst I fragments of 1.3 kb and 1.25 kb are marked
with (•) near position 235. The extent and location of selected cosmid
clones are shown beneath the restriction map. The ends of cosmid Inserts
(•) and their internal Sal I sites (I) are indicated. A 6 kb repeat
sequence Is represented by a heavy arrow at 0-6 kb and 93-100 kb. Its
orientation Is arbitrary. The approximate positions of rRNA and proteincoding genes (Table 1) are given above the map.
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Nucleic Acids Research
S P S P S P
P16 7 P11-6
P10-5,
S644.
S17,
A
P C
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K
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5656
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SX
Nucleic Acids Research
(Fig. 1A, lanes S and K). Because of the large (>50) number of Pst I
fragments, some ambiguities remain in t h e i r locations.
A prominent
s t r u c t u r a l f e a t u r e of the genome i s a two-copy d i r e c t r e p e a t of
approximately 6 kb that contains part or a l l of the 26S rRNA gene (Fig. 3, 0
and 93 kb; ref. 24). This is the only major repeated sequence that is
detectable by f i l t e r hybridization.
Evidence that the "6 kb repeat" is
involved in the generation of a t r i p a r t i t e genome organization is given in
the next section.
Intramolecular Recombination In Spinach mtDNA
The 6 kb repeat possess a single Sal I s i t e , and is contained within
Sal I fragments of 1.7 kb, 19.5 kb, 17.1 kb and 6.44 kb (Fig. 3). The 1.7
kb and 17.1 kb Sal I fragments and the 19.5 kb and 6.44 kb Sal I fragments
cross-hybridize, since each pair shares common repeat sequences (Fig. 3 and
Figs 4B and 4C, lanes S). Each fragment also cross-hybridizes with Pst I
fragments of 17.5 kb, 16.7 kb, 11.6 kb and 10.5 kb (Figs. 4B and 4C, lanes
P).
The four repeat-containing Pst I fragments are substoichiometric, as
judged by their reduced fluorescence relative to other Pst I fragments, in
an ethldlum bromide-stained gel (Fig. 4A, lane P). Of these submolar Pst I
fragments, only those of 17.5 kb and 10.5 kb are accounted for by the
r e s t r i c t i o n map shown in Fig. 3. The 16.7 kb and 11.6 kb Pst I fragments,
however, can be generated by a single postulated recombination event between
the repeat sequences embedded within the 17.5 kb and 10.5 kb fragments. The
s t r u c t u r a l r e l a t i o n s h i p s of the four Pst I fragments,
the larger,
substoichiometric Kpn I fragments within which they l i e , and the four
repeat-containing Sal I fragments are shown in Fig. 4D. Each pair of Kpn I
(or Pst I) fragments conserves genome size (i.e. for Kpn I, 25 + 43 kb - 48
+ 20 kb), but contain different combinations of flanking sequences.
The f i l t e r h y b r i d i z a t i o n data shown in Figs. 4B and 4C, the
substoichiometric levels of the four repeat-containing Pst I fragments (Fig.
4A, lane P), and their physical relationships (Fig. 4D), are consistent with
Figure 4. Filter hybridization analysis of the 6 kb repeat. Spinach mtDNA
was digested with Sal I (S) and Pst I (P), electrophoresed in a 0.7% agarose
gel, stained with ethidium bromide (A), transferred bidirectionally (ref.
51) to GeneScreen and hybridized with 32 P-labelled pS6.44 (B) and pS1.7 (C).
Sizes of fragments are in kb, and were determined as in Fig. 1. (D)
Organization of the four repeat-containing Kpn I fragments. K25 and K43 are
located on the "master genome" (Figs. 3 and 5A), and K48 and K20 are found
on the 93 kb and 234 kb c i r c l e s , respectively (Fig. 5C). The two pairs of
fragments (K25/K43 and K48/K20) are postulated to Interconvert via
reciprocal recombination (Fig. 5B). The locations of the four repeat
containing Pst I fragments (P10.5, P17.5, P16.7 and P11.6; Figs. 4A-C) are
indicated by thickened l i n e s . Sal I fragments discussed in the text are
SI.7, S19.5, S17.1 and S6.44. Selected r e s t r i c t i o n s i t e s are shown for Kpn
I (K), Pst I (P), Sal I (S), Eco RI (E) and Bgl II (G). The maximum extent
of the repeat is shovn at the top by an open box; The f i l l e d portion
represents i t s minimum size.
The orders of the two Pst I fragments
completely internal to S19.5, and of those to the left of S17.1 are unknown.
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Nucleic Acids Research
B
Figure 5. (A) Restriction map of the spinach mtDNA master genome. Pst I
(inner circle), Sal I (middle circle) and Kpn I (outer circle) fragments are
shown. The 6 kb repeat is indicated by heavy arrows. The portions of the
naster genome from which the 93 kb and 234 kb subgenomes are derived are
shown as broken and solid lines, respectively, on the innermost circle. A
postulated reversible recombination event is diagrammed in (B), leading to
the formation of two subgenoraes via dissolution of the master genome (C), or
to cointegration of the two smaller circles to form the master genome (A).
the occurrence of intragenomic recombination.
The 6 kb repeats present in
d i r e c t o r i e n t a t i o n on the 327 kb "master genome" (Fig. 5A) are proposed to
pair (Fig. 5B), leading to recombination and the resolution of two
"subgenomes" of 93 kb and 234 kb (Fig. 5C). Presumably, t h i s event is
reversible via cointegration of the two smaller circles to form a "master"
genome. Furthermore, either of the smaller circles could recombine with the
naster genome, leading to c i r c l e s of 420 kb and 561 kb. These molecules
could be involved in further events, ad Infinlcum. We hypothesize, however,
that the major molecular forms of the genome are those shown in Figs. 5A and
5C. An exact evaluation of the r e l a t i v e proportions of the master genome
and two subgenomes is difficult.
Nonetheless, i t Is clear that the repeatcontaining Sal I fragment of 1.7 kb gives a hybridization signal that is
approximately two-fold stronger with the 16.7 kb Pst I fragment than with
the 17.5 kb Pst I fragment, although i t has an equal length of homology with
each (Fig. 4D). There is a less marked, but correspondingly stronger signal
with the 11.6 kb Pet I fragment compared to the 10.5 kb Pst I fragment (Fig.
AC, lane P). These r e s u l t s together suggest that the subgenomes, which
contain 16.7 kb and 11.6 kb Pst I fragments (Fig. 5C), are present in higher
abundance than the 327 kb master genome, which contains 17.5 kb and 10.5 kb
Pst I fragments (Fig. 5A). We emphasize, however, that these estimates are
hampered by d i f f e r e n t i a l degradation of fragments, and the d i f f i c u l t y of
resolving the 17.5 kb and 16.7 kb Pst I fragments s u f f i c i e n t l y to perform
densltometrlc scans.
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Nucleic Acids Research
TBbla 1.
Mapping of Spinach Mitochondrial Genes
Gene*
Clone
26S
pS4.8
18S
5S
Source
Species Vector
Insert
Size
(to)
Hybridising spinach mtOft frat»nants (to)
Sal I
Pat I
l^n I
Eraymo
KBf.
Sna I
1
19.5, 17.1,
6.44, 1.7
17.5,
11.6,
16.7,
10.5
48, 43,
25, 20
com
PUC8
pP3.2
oom
pUC8
3.2
Pot I
2
36
4.0
4.5
206NX
com
pUCL2
0.085
Mml-Hjpall
**
36
4.0
4.5
cob
pEl.5
Oenothara
1.5
EooRI
50
24
9.6
38.5
pBR322
4.8
CCKl
PEE3.9
com
pUC8
3.9
BnHI-EcoRI
4
12
OOX2
peMa
com
DER322
2.4
Bcota
5
24, 5.45
urf-1
pm.9
watermelon
pUC8
1.9
Hindm
10
12
28
28, 13.5
at!*
pH2.7
Oulll
pocu
2.7
Hindm
8
atpe
FH4.2
<.t II II
PUC19
4.2
Hindm
6
11.4
38.5
7.6
43, 20
5.4
48, 43
5.1, 3.6
15
24.5
19.3
Gene designations are: 26S, 18S, 5S; genes encoding RA components of the witnriiuili Inl
ribosaras; cob, apocytochrome B; ooxl and oox2, subunits I and n of cytochrome axidase,* urf-1,
gene homologous to unidentified reading frame-1 of animal and fungal mitochondria (11-14); atpft
and atp6; alpha subunit of the Fj-MFase ana oubunit 6 of the F -KTPaaa, rsBpectiviely.
A. Itoloney, Dqnrtnent of Biological Scienaea, Stanford University.
Mapping of Mitochondrlal Genes
To p l a c e known m i t o c h o n d r i a l genes on the p h y s i c a l map of spinach
mtDNA, p r e v i o u s l y c h a r a c t e r i z e d g e n e - c o n t a i n i n g c l o n e s were used in
heterologous f i l t e r hybridizations. A description of the clones, and a l i s t
of hybridizing fragments i s given in Table 1. Sample f i l t e r hybridizations
with gene-containing clones for 18S rRNA, apocytochrome B and subunit II of
cytochrome o x l d a s e are shown i n Fig. 6. Host of the genes are s c a t t e r e d
widely throughout much of the spinach mitochondrial genome (Fig. 3). A sole
exception to t h i s pattern i s the close linkage of the 18S and 5S rRNA genes,
which are also adjacent in other plant mitochondrial genomes (1, 36).
All
the genes are p r e s e n t only once per genome, except for the 26S rRNA gene,
which, as p r e v i o u s l y noted (24), i s c o i n c i d e n t w i t h the 6 kb repeat and
e x i s t s in two copies per genome.
ReRions Homologous with Chloroplast DNA
It has previously been established that substantial sequence homologies
e x i s t between ctDNA and mtDNA in flowering plants, including spinach (20,
37). To further our understanding of the nature of these i n t e r o r g a n e l l a r
DNA h o m o l o g i e s , r e g i o n s of homology between spinach c h l o r o p l a s t and
mitochondrial DNAs were mapped by f i l t e r h y b r i d i z a t i o n . Pst I , Sal I and
Kpn I r e s t r i c t i o n d i g e s t s of mitochondrial and c h l o r o p l a s t DNAs were
electrophoresed in agarose g e l s , transferred to GeneScreen, and probed with
P-labelled clones of spinach ctDNA. Typical r e s u l t s are shown in Fig. 7.
A cloned spinach ctDNA Pst I fragment of 1.9 kb, PI.9, h y b r i d i z e s as
expected to a 1.9 kb fragment p r e s e n t both i n the ctDNA lane and (as a
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Nucleic Acids Research
P S K P S K P S K
36
*
»
9-6
401
18S
COB COM
Figure 6. Mapping of rRNA and protein-coding genes. Spinach mtDNA digested
with Pst I (P), Sal I (S) or Kpn I (K) was electrophoresed in 0.7% agarose
g e l s , t r a n s f e r r e d to GeneScreen, and hybridized with the Indicated gene
clones (cf. Table 1).
contaminant) in the mtDNA p r e p a r a t i o n [Fig. 7, PI.9, lanes P(c) and P(m)].
The clone a l s o hybridizes with a bona fide mtDNA Pst I fragment of 3.6 kb,
which i s not present in the ctDNA (38). Sequences present in PI.9 are
therefore homologous to sequences that l i e within the mltochondrial 3.6 kb
Pst I fragment.
Similar h y b r i d i z a t i o n s with Sal I, Kpn I and doublydigested mitochondrial and c h l o r o p l a s t DNAs (Fig. 7; double d i g e s t s not
shown), allow placement of the homologous region within a 3.6 kb Pst I
fragment t h a t l i e s within a 12 kb Sal I fragment (cf. Figs. 3 and 8). A
more complex pattern is obtained with a cloned spinach ctDNA Xho I fragment
of 15.3 kb (Fig. 7, X15.3). This fragment contains the chloroplast 16S rRNA
gene (39); i t s weak hybridization with the mltochondrial 18S rRNA gene (Fig.
7, X15.3, open arrows) is therefore expected (40). X15.3 also hybridizes
with two other d i s t i n c t regions of the mitochondrial genome (Fig. 7, X15.3,
f i l l e d arrows).
Fine mapping of these homologies was accomplished for clones spanning
v i r t u a l l y the e n t i r e spinach c h l o r o p l a s t genome. The regions of crosshybridization are Indicated schematically in Fig. 8, and are summarised in
Table 2.
At l e a s t 14 regions of spinach mtDNA are homologous with
chloroplast DNA. Two of these homologies result from the primordial common
ancestry of the mitochondrial and c h l o r o p l a s t rRNA genes (Fig. 8, dotted
lines).
Whether any of the 12 other ctDNA-homologous mtDNA sequences
contain functional genes i s currently unknown.
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P
S
K
P
S K
P
S K
m c mc m c
P1-9
X15-3
Figure 7. Homologies between spinach mitochondrial and chloroplast DNAs.
Mitochondrial (m) and chloroplast (c) DNAs were digested with Pst I (P), Sal
I (S) or Kpn I (K), electrophoresed in a 0.7% agarose g e l , s t a i n e d with
ethldium bromide (left), transferred to GeneScreen, and hybridized with the
nick-translated ctDNA clones indicated under each panel. MtDNA r e s t r i c t i o n
fragments homologous with the ctDNA probes are marked with closed arrows
(^). Open arrows « ) designate mtDNA fragments of 4.0 kb (Pst I ) , 4.5 kb
(Kpn I) and 36 kb (Sal I) which contain the 18S rRNA gene, and are therefore
presumed (40) to c r o s s - h y b r i d i z e with the 16S rRNA gene contained within
X15.3. The f i l t e r probed with X15.3 was derived from a d i f f e r e n t gel than
the one shown, and does not a l i g n p r e c i s e l y with the s i z e markers on the
left.
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ctCNA M j
E3«I
twl
ISl-a kb
mtDNA - Sal I
Figure 8.
Schematic r e p r e s e n t a t i o n of homologies between spinach
mitochondrlal and chloroplast DNAs. Restriction maps of the chloroplast and
mitochondrial genomes are drawn to the same scale. Solid lines are drawn
between ctDNA clones and the mtDNA r e s t r i c t i o n fragments to which they
hybridize (see Fig. 7 and Table 2). The dotted lines Indicate homologies
observed as a consequence of hybridization between chloroplast and
mitochondrial rRNA genes. Arrows above the ctDNA restriction map, and below
the mtDNA map, represent the ctDNA inverted repeat and the mtDNA 6 kb
repeat, r e s p e c t i v e l y . "16" and "23" are the approximate p o s i t i o n s of the
c h l o r o p l a s t genes for 16S and 23S rRNA. 26S and 18S rRNA genes are shown
below the mtDNA map. Only one of the two 26S rRNA genes i s shown and only
one copy of the mtDNA 6 kb repeat i s pictured as hybridizing with X3.0.
Similarly, cross-hybridizations, and the chloroplast rRNA genes, are shown
for only one segment of the ctDNA inverted repeat.
DISCUSSION
Plant Mitochondrial Genome Structure
Our mapping studies p r e d i c t a t r i p a r t i t e s t r u c t u r e for the spinach
mitochondrial genome. A 327 kb "master genome" and two subgenomes of 93 kb
and 234 kb are interconverted by reciprocal recombination within a 6 kb
direct repeat. The t r i p a r t i t e organization of spinach mtDNA parallels that
f i r s t reported for turnip mtDNA, which contains a 218 kb master genome and
subgenomes of 135 kb and 83 kb (27). Furthermore, four species in Brasslca
and the closely related genus Raphanus feature a similar t r i p a r t i t e mtDNA
organization (J. Palmer and L. Herbon, unpublished data). In each case, the
recombination repeats are in direct orientation, despite the occurrence of
multiple Inversions throughout the genome.
The organization of spinach and several Brass ica and Raphanus
nltochondrial genomes contrasts with that of corn and wheat mtDNAs. Corn
mtDNA is postulated to have a highly complex multipartite organization, in
which five p a i r s of repeats are actively engaged in recombination (26).
Similarly, wheat mtDNA possesses multiple pairs of repeated and recombining
sequences (41), although a complete physical map is unknown.
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Table 2. Cross-Hanologies Between Spinach ntOA and Cloned spinach chloraplast DA.
Hybridizing
Hybridizing
mtna fiauiuails***
ctafK fragments**
Pst I
Kpn ][
Clone*
Sal I
Sal I
Pst I
mm i
P7.7
7.7
22.3
22.3
9.7, 1 . 9
17.5
16.7
17.1
48
43
P12.3
12.3
27.3
20.5
9.7
4.4, 1 . 6
17.5
13.3
10.3
6.44, 5.45
NT
P25.5
25.5
20.5, 5.2
6.0, 4.0
1.6, 18.4
3.2, 26.6
47.9
36.9
26.6
X3.0
53.7
0.80
6.5
17.5+, 11.6
16.7+, 10.5
3.6
19.5 + , 6.44+
6.40
NT
NT
X12.8
53.7
47.9
26.6
4.6
X11.9
53.7
47.9
26.6
4.2
XP1O
53.7
10.6, 47.9
36.9
6.8, 17.5
4.0 , 16.7
PX5.1
8.9
10.6
36.9
11.4
XP3.8
8.9
10.6, 9.0
36.9
13.3, 7.45
P17.3
17.3
9.0
13.9, 2.4
36.9, 22.3
ncne
none
ncra
PI.9
1.9
13.9
22.3
3.6
12
43
P13.5
13.5
22.3, 13.9
22.3
4.85
22.2
6.0
22.2
3.7, 17.1
24
2.80, 19.5
15.5
ia.4
12.5, 4.8
4.5 + , 43
38.5
NT
a c n e s (ref. 35) are designated by enzyme (P - Pst 1, X - Xho I) and length of insert in kb.
Fragoents hybridizing with XP10, PXS.l and XP3.8 ware determined by occpnring r e s u l t s using
overlapping 15.3 kb Xho 1 and 8.9 kb Pst 1 clones.
** Fragrant s i z e s are in kb. Happing data are from r e t s . 38 and 39.
*** Ftajueiit s i z e s are in kb. NT - not tested.
+
Hybridization resulting fran horology between nltcchondrial and chloroplast rtUAs.
Several trends are emerging in the str uc tur e of plant mtDNAs. Most
genomes are characterised by the presence of large repeated sequences. Host
of these repeats are Involved In i n t r a - and Intergenomlc recombination.
Such "recombination repeats" have been reported for turnip (27), corn (26),
wheat (24, 41) and pokeweed mtDNAs (24), in addition to spinach mtDNA.
Where It has been determined, a l l recombination repeats are direct repeats.
Accordingly, a l l f u l l y mapped p l a n t m 1 tochondrial genomes have a
m u l t i p a r t i t e o r g a n i z a t i o n , as opposed to the f l i p - f l o p
dualism
c h a r a c t e r i s t i c of inverted repeat-containing chloroplast genomes (42).
Finally, the monocots examined thus far, corn and wheat, feature multiple
pairs of repeated sequences, whereas spinach and five species of Brass lea
and Raphanus possess only a single major repeat. We note that a l l the above
observations are based on an extremely limited sample size.
For example,
the much larger cucurbit mitochondrial genomes (43) may well have a more
complex structure than the smaller dicot genomes.
Organization of Mitochondrial Genes
The map positions of six protein and three rRNA-encodlng genes have
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Nucleic Acids Research
been deternlned by heterologous f i l t e r hybridizations (Figs. 3 and 6).
These genes are scattered throughout the genome. Given the small(15-25,
ref. 23) number of proteins thought to be encoded by plant mitochondria, and
t h e i r large genome sizes, the p o s s i b i l i t y has been raised (23) that
functional genes may be clustered, with the c l u s t e r s separated by long
regions of non-coding DNA. Yeast mtDNA, for example, contains long,
presunably non-coding, (A+T)-rich regions (44). Our findings for spinach do
not suggest any clustering, or "operon-like" regions, with the exception of
the 18S and 5S rRNA genes, which are closely linked, as in a l l plant
mitochondrial genomes examined (1, 36) (Fig. 3). Many more genes remain to
be mapped, however, and i t is s t i l l possible that c l u s t e r i n g occurs for
other loci.
Homology with Chloroplast DNA
I t has been previously established that sequence homologies exist
between chloroplast and mitochondrial DNAs in a wide variety of flowering
p l a n t s (20, 21, 45). I t was found that v i r t u a l l y every ctDNA clone tested
hybridizes with one or more plant mtDNAs, and that in the case of spinach,
five mung bean ctDNA clones hybridize with mtDNA (20). Cross-homology
between a 7.7 kb spinach ctDNA Pst I fragment and spinach mtDNA has been
more fully characterized (37). We have mapped t h i s homology to a region
near the end of the mtDNA 6 kb repeat (Fig. 8).
Furthermore, we have
extended these mapping studies by u t i l i z i n g spinach ctDNA clones spanning
nost of the genome, and by locating the positions of homologous sequences on
the mtDNA restriction map.
Eleven of the twelve ctDNA clones tested hybridize with mtDNA (Table
2).
As in the case of bona fide
mitochondrial genes (Fig. 3), regions
homologous with ctDNA are highly dispersed in the mitochondrial genome (Fig.
8).
In addition, the extent of homology, as Judged by the relative strength
of cross-hybridization signals, is quite variable from one region to another
[e.g.
Fig. 7, lane X15.3 P(m)]. Because both the length and degree of
complementarity of these homologous sequences are not known, we cannot
speculate on the timing of i n t e r o r g a n e l l a r DNA transfer.
I t is obvious,
however, t h a t the linear order of cross-hybridizing sequences is quite
d i f f e r e n t in the two genomes (Fig. 8).
This suggests e i t h e r that the
n i t o c h o n d r i a l genome has undergone m u l t i p l e rearrangements since
Interorganellar DNA transfer occurred, or that many separate events have led
to d i s p e r s a l of t h i s "promiscuous" DNA. We feel i t l i k e l y that both
multiple transfer events and genome rearrangements have contributed towards
the present s i t u a t i o n .
Further studies, Including nucleotide sequence
analysis, are required to refine our knowledge of the frequency, direction
and mechanism of DNA exchange between organelles.
These ctDNA-homologous sequences, along with bona fide mitochondrial
genes and repeated sequences engaged in recombination, comprise less than
one third of the 327 kb spinach mitochondrial genome. The origin, function
and composition of the remainder Is currently unknown. One p o s s i b i l i t y ,
proven so far for corn (46) and spinach (37), as well as for animals (47,
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Nucleic Acids Research
48) and fungi (49) is that nuclear DNA is homologous with mtDNA. While the
extent of these homologies is unclear, a physical map and clone bank of the
mitochondrial genome w i l l f a c i l i t a t e t h e i r analysis and also aid in
unraveling the mode of plant mitochondrial gene expression.
ACKNOWLEDGMENTS
We thank William Thompson for the laboratory f a c i l i t i e s used to perform
t h i s work. D.B.S. was s u p p o r t e d by an N.I.H. t r a i n i n g g r a n t and a McKnight
I n t e r d i s c i p l i n a r y Award in Plant Biology. This i s CIW-DPB Publication #924.
*To whom correspondence should be addressed
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