Plant Molecular Biology 16: 593-600, 1991.

© 1991 Kluwer Academic Publishers. Printed in Belgium. 593

Isolation, physical map and gene map of mitochondrial DNA from the
cryptomonad Pyrenomonas salina

Martina Maerz and Peter Sitte

Institut fiir Biologie II, Lehrstuhl fiir Zellbiologie, Schanzlestr. 1, D-7800 Freiburg, Germany

Received 8 August 1990; accepted in revised form 29 November 1990

Key words: cryptomonads, gene map, mtDNA, mitochondrial evolution, Pyrenomonas salina, restriction
map

Abstract

Mitochondrial D N A (mtDNA) from the cryptomonad Pyrenomonas salina was isolated by CsCl-buoyant
density centrifugation of whole-cell D N A in the presence of Hoechst dye 33258. m t D N A consists of
circular molecules about 47 kb in size as estimated from restriction enzyme analysis. A physical map for
six restriction enzymes (Bam HI, Bge I, Eco RI, Pst I, Sac I and Sal I) has been constructed. Genes coding
for the small subunit ofrRNA, cytochrome oxidase subunits I and II, and apocytochrome b were localized
on this map using Southern blot hybridization with heterologous gene probes from Oenothera. Genes for
5S rRNA and N A D H dehydrogenase subunit 5 are absent from P. salina mtDNA. The mitochondrial
genome, being the first analysed to this extent in chromophytic algae, should be valuable for taxonomic
and phylogenetic studies.

Introduction geneous mixtures of circular and linear plasmids

Mitochondria are semiautonomous organelles
that contain their own DNA. Although in virtu-
ally all eukaryotic cells their function in oxidative
phosphorylation is the same, mitochondrial gen-
omes of animals, plants, fungi and unicellular
eukaryotes such as protists and algae show a wide
diversity in genome size, structure and gene
arrangement. Whereas most vertebrate mito-
chondrial genomes are circular monomers of
about 16 kb [45], mitochondrial DNAs of fungi
vary in size from 18.9kb to 176.3 kb [8, 27].
Differences in the organization of sequence occur
and intervening sequences, not found in animal
mtDNAs, are common. In higher plants mito-
chondrial genome size range not only from 200 kb
to 2400 kb but genomes can also be hetero-
and circular main molecules. Within the protists
mitochondrial genomes exist in three alternative
forms: closed circular (most common), linear and
aggregates of large and small circles [39].
However, in all systems studied the informa-
tion coded by the mitochondrial genomes seems
to be remarkably conserved. It consists of genes
for the small and the large subunit of rRNA, a set
of mitochondrial tRNAs and several proteins
which are important components of the inner
mitochondrial membrane.
Whereas the mtDNAs of animals, higher plants
and fungi are well studied, only few data exist of
mitochondrial genomes from protists and algae.
Published data of algal mtDNAs are restricted
to the chlorophytes and euglenoids, whose chloro-
plasts contain chlorophylls a and b. We have
594
investigated the m t D N A of the cryptomonad alga
Pyrenomonas salina, a member of the chromo-
phytic lineage (chlorophylls a and c).
Cryptomonads are of special interest for testing
the serial endosymbiont theory (SET [44]).
According to this theory, the uptake of symbionts
and their reduction to plastids has not been a
unique event but occurred several times leading to
the modern green algae, red algae and glauco-
cystophytes. Moreover, not only prokaryotic
organisms served as symbionts but also
eukaryotic cells. Endocytic eukaryotes are sup-
posed to be the progenitors of all chloroplasts
surrounded by more than two membranes (e.g.
euglenoids, chromophytes, dinoflagellates, cryp-
tomonads [ 18, 46]). Cryptomonads display either
a lasting intermediate state of reduction of an
endosymbiotic eukaryotic cell to an organelle, or
a 'young' form of symbiosis where this reduction
is still incomplete. Cryptomonad chloroplasts are
surrounded by four membranes. The outer pair,
the so-called periplastidal ER [ 17], surrounds the
former symbiont which in turn consists of a
chloroplast surrounded by two membranes of the
chloroplast envelope, starch grains, small
amounts of cytoplasm with some 80S-type ribo-
somes, and the remnants of the former nucleus of
the symbiont, the nucleomorph. Dictiosomes and
mitochondria are entirely absent from the
'symbiont' [40].
The symbiont is supposed to be related to red
algae due to pigments of the chloroplast and
features diplayed by the chloroplast genome
[12, 19]. However, almost nothing is known
about the former host. As organellar DNAs have
proved to be a useful tool for studying phylo-
genetic relationships, we have characterized the
mitochondrial genome from Pyrenomonas salina
to elucidate the phylogenetic descent of the host
cell.
Materials and methods
P. salina cells were grown at 20 ° C in f/2 medium
[22] on a 12 h light/12 h dark cycle.
Cells were harvested in late logarithmic phase
by centrifugation at 1000 × g for 10 min at 4 °C
and washed twice with f/2. The final pellet was
resuspended in 4 ml/g wet weight isolation buffer
( 5 0 m M Tris, pH 9.0, 100mM Na2 EDTA,
150 m M NaC1). Cells were lysed by adding SDS,
sodium-desoxycholate and predigested Pronase
(BOhringer, Mannheim) to final concentrations of
0.5 ~ , 1.0~ and 1 mg/ml, respectively. The lysate
was kept at 4 oC overnight. After extracting twice
with phenol and once with chloroform/isoamyl
alcohol (24: 1), the aqeuous phase was dialysed
against 1 x SSC (150 m M NaC1, 15 mM sodium
citrate), 0.5 mM Na 2 EDTA. 1.1 g solid CsC1/ml
D N A solution was gently dissolved, sodium-
sarkosyl and Hoechst dye 33258 were added to
final concentrations of 0.1 ~o and 4 #g/~g DNA,
respectively. Gradients were run in a Ti 50 rotor
(Beckman) at 37000 rpm and 18 °C for 20 h.
m t D N A bands at a lower density than plastid and
nuclear DNA, consistent with a content of 33.7 ~o
G + C, compared with 38.8~ G + C for plastid
and 60.2~o G + C for nuclear D N A [23].
mtDNA-containing bands were removed under
long-wave UV illumination and afterwards
dialysed against TE (10 m M Tris-HCl, pH 8.0,
1 mM Na2 EDTA). D N A solution was made
0.4 M in LiC12 and precipitated with two volumes
of EtOH. D N A was finally collected and dis-
solved in TE at concentrations of 0.1 #g//~l.
Restriction enzymes were purchased from
varied sources and digestions were carried out as
recommended by the suppliers.
For construction of the physical map the
double-digestion method [24] was used.
D N A fragments seperated in 0.6-1.0 ~ agarose
gels were transferred to Nylon membranes by
vacuum blotting (LKB 2016 VacuGene).
D N A to be used as probes was produced
according to Maniatis et al. [32] and labelled with
Biotin-16-dUTP by the random primer method
[14].
Hybridizations were carried out according to
Gebeyehu et al. [ 16], but buffers for prehybridi-
zation and hybridization were used without the
addition of formamide. Filters were washed in
2× SSC, 0.1~o SDS (2 × 5min, at room tem-
perature) and 0.5 x SSC, 0.1~o SDS
 595

(2 x 15 min, at 60 “C) for high-stringencycondi- Results

tions or 1 x SSC, 0.1% SDS (2 x 15 min, at
50 “C) for low-stringency conditions.
Calorimetric detection of biotinylated DNA
probeswas carried out with a detectionkit (Gibco
BRL) as recommended.
Isolation of mtDNA
The ultrastructure of P. salina, and the unique
network-like mitochondrion in particular [ 371,
made it impossible to isolate intact organelles.
However, extraction and recovery of whole-cell
DNA has beensuccessfulfor isolation of mtDNA

Table I. Summary of Pyrenomonassalina mtDNA sizes (in kb) obtained from restriction enzyme analysis. Origins of fragments
generated by double digestions are given in brackets. Abbreviations used: B, Bum HI; Bg, Bgl I; E, Eco RI; P, Pst I; S, Sal I;
Sa, Sac I.

Fragment Sal I Pst I Bum HI Eco RI Bgl I/&Z I BgZI/Bum HI Pst I/Sal I Bum HI/Sac I

1 34.5 17.5 33.6 12.8 34.5 (Bgl, Sl) 32.0 (Bgl, Bl) 16.8(Pl, Sl) 33.0 (Bl, Sal)
2 12.4 16.8 7.6 11.4 6.9 (Bgl, S2) 7.4 (Bgl, B2) 14.9 (P2, Sl) 6.6 (B2, Sal)
3 8.6 3.6 5.4 6.0 (Bgl, S2) 3.6 (Bgl, B3) 6.4 (P3, Sl) 3.6 (B3, Sal)
4 3.5 1.4 4.2 2.1 (Bgl, Bl) 3.1 (P4, S2) 1.4 (B4, Sal)
5 1.0 3.6 1.4 (Bgl, B4) 2.9 (P2, S2) 1.2 (B2, Sal)
6 3.2 1.0 (Bgl, B5) 1.9 (P3, Sl) 1.0 (B5, Sal)
I 1.8
8 1.6
9 1.3
10 1.0

Sum of
fragment
sizes 46.9 46.4 41.2 46.3 41.4 41.5 46.4 46.8

Fragment Bum HI/&Z I Bum HI/Pst I Sal I/Eco RI Pst IIEco RI Bum HI/Eco RI

1 25.7 (Bl, Sl) 16.8 (Bl, Pl) 12.3 (Sl, El) 11.72” (Pl, E2, P2, E3) 11.4 (Bl, Ell)
2 7.9 (Bl, S2) 8.6 (Bl, P3) 11.4 (Sl, E2) 5.5 (P2, E3) 7.5 (Bl, El)
3 7.6 (B2, S 1) 7.6 (B2, P2) 3.5 (S2, E5) 4.3 (P3, E4) 4.8 (B2, El)
4 3.1 (B3, S2) 6.2 (B 1, P2) 3.2 (Sl, E6) 3.4 (P3, E5) 4.2 (B 1, E4)
5 1.4 (B4, S2) 3.0 (B3, P2) 3.1’” (S2, E4, Sl, E3) 3.2 (Pl, E5) 3.6 (B 1, E4)
6 1.0 (B5, Sl) 1.6 (Bl, P4) 2.6 (S2, E3) 1.8 (P4, E7) 3.2 (Bl, E6)
I 0.5 (B3, S 1) 1.4 (B4, P4) 1.8 (S2, E7) 1.2 (P4, E9) 3.1 (B3, E3)
8 1.0 (B5, P2) 1.6 (Sl, E8) 1.1 (P3, E8) 2.7 (B2, E3)
9 0.6 (B3, P4) 1.2 (S2, E9) 1.0 (Pl, El) 1.6 (Bl, E8)
10 1.0 (S2, ElO) 0.8 (P4, ElO) 1.2 (B4, E9)
11 0.8 (Sl, E4) 0.7 (Pl, El) 1.1 (Bl, E9)
12 0.3 (P2, E7) 0.9 (B5, El)
13 0.2 (P4, E9) 0.5 (B3, E7)
14 0.3 (B4, ElO)
15 0.2 (Bl, ElO)

Sum of
fragment
sizes 41.2 46.8 45.6 46.9 46.7
596

from P. salina. The three distinct bands achieved
in CsCI buoyant density centrifugation of whole-
cell D N A [23] could be further separated by
adding Hoechst dye 33258. This method made it
possible to remove m t D N A bands from the
gradients without or with only minor contami-
nations of plastid D N A that bands beyond
mtDNA.
two fragments, digestion with Pst I led to four
fragments, whereas Bam HI generated five and
Eco RI ten fragments. A summary in Table 1 and
Fig. 1, single digests with restriction enzymes
Barn HI, Eco RI and Pst I, and double digests
with restriction enzyme pairs Bam HI/Eco RI,
PstI/Eco RI and Eco R I / S a l I resulted in a
P. salina mt genome size of 46.8 + 0.5 kb.

Restriction enzyme analysis of mtDNA

P. salina m t D N A was digested with restriction
enzymes Bam HI, Bgl I, Eco RI, Pst I, Sac I, and
Sal I to determine mitochondrial genome size and
for construction of a physical map. Unique sites
were found for Bgl I and Sac I. Sal I generated
Construction of a physical map of P. salina mtDNA
A physical map for P. salina m t D N A was con-
structed according to Herrmann et al. [24]. The
m t D N A was digested with one restriction
enzyme, and the fragments generated were sub-

Fig. 1. Restriction endonuclease digestion ofmtDNA from P. salina. A, Hind III-digested 2-DNA; B, Eco RI; C, Eco RI/Pst I;
D, Pst I; E, Hind Ill-digested 2-DNA; F, Sail; G, SalI/Eco RI; H, Eco RI; I, Barn HI; J, Bam HI/Eco RI. (Minor bands in
background: plastid DNA.)

Fig. 3. Southern blots showing hybridization of biotinylated mtDNA from Oenothera. Filters have been hybridized with genes I~
for rRNA (lane A'), cytochrome oxidase subunit I (lane B', C'), cytochrome oxidase subunit II (lane D') and apocytochrome
b (E'). Digestions have been generated with: A, Eco RI/Pst I; B, Eco RI; C, Eco RI/Pst I; D, Barn HI; E, Eco RI/Sal I.
 597

rrnA COil Table2. Hybridization of mitochondria and chloroplast-

specific genes to P. salina mtDNA.

Probes Stringency Probe source Reference

COl high Oenothera(mt) 26

COIl high Oenothera(mt) 25
CYB high Oenothera(rot) 37
rrnA (18S) high Oenothera(mt) 47
rrnA (5S) low Oenothera(mt) 47
ND5 low Oenothera(mt) 47
rbcL low Spinach chloroplast 49
psbA low Spinach chloroplast 2

sequently separated by low-gelling-temperature

Fig. 2. Physical map of the circular mtDNA from P. salina.
Fragment numbers present in the map were assigned in
Table 2. Approximate locations of rrnA, CO1, COII, and
CYB are given.
agarose gel electrophoresis. Each fragment was
excised out of the gel, digested with a second
restriction enzyme and again subjected to gel elec-
trophoresis. Using the same method, fragments
generated with the second restriction enzyme
were digested with the first one used in the former
experiment. Such double digestions were carried
out using restriction enzyme pairs Pst I/Eco RI,
Bam HI/Eco RI, and Psi I/Sal I. The distances
598
between the unique sites of Bgl I and Sac I were
then determined by combination of double and
triple digests and finally could be ordered as
shown in Fig. 2 and Table 1.
Gene localization
To verify the mitochondrial origin of the received
molecule and to get further information about
gene location in cryptomonad m t D N A random
primed heterologous D N A probes from
Oenothera were hybridized to Southern blots of
P. salina mtDNA. Hybridization conditions and
probe sources are presented in Table 2. Results
are shown in Fig. 2 and 3.
An r D N A probe (rrnA) containing the 18S
r D N A of Oenothera hybridized to fragment PE 6,
which has an estimated length of 1.6 kb. This
observation suggests that P. salina m t D N A
contains a unique set o f r R N A genes. An apocyto-
chrome b probe (CYB) hybridized to fragment
BE 8, a cytochrome oxidase subunit I probe
(CO1) to E 4 and E 5, and a cytochrome oxidase
subunit II probe (COIl) to BE 7. A 5S rRNA
(rrnA) gene probe and a probe for subunit 5 of
N A D H dehydrogenase (ND5) from Oenothera as
well as gene probes for the large subunit of
ribulose- 1, 5-biphosphate carboxylase (rbcL) and
32 kDa protein (psbA) from spinach chloroplast
D N A gave no signals (data not shown).
Discussion
Pyrenomonas salina m t D N A consists of circular
molecules with a size of about 47 kb. This data
correspond well to those found by electron micro-
scopic analysis [23].
Gene probes for the small subunit ofrRNA, 5S
rRNA, apocytochrome b, NADH-dehydro-
genase subunit 5, and cytochrome oxidase sub-
units I and II from Oenothera as well as gene
probes for the large subunit of ribulose-1, 5-bis-
phosphate carboxylase and 32 kDA protein from
spinach chloroplasts were hybridized against
restriction fragments of m t D N A from P. salina.
Most genes of mitochondrial origin could be
localized on the genome whereas those of plastid
origin could not.
The lack of a 5S rRNA gene is not surprising,
because 5S rRNA genes are absent in mito-
chondrial genomes other than those of plants
[42]. Genes for some subunits of the NADH-
dehydrogenase complex have been identified in
the m t D N A of mammals [4,6], Drosophila
[9, 10], higher plants [43, 47], Chlamydomonas
[5], Aspergillus [21], and Neurospora [28] as well
as in the maxicircle D N A oftrypanosomatid pro-
tozoa [ 11 ]. However, m t D N A of Schizosaccharo-
mycespombe [ 29] and Torulopsis glabrata [ 7 ] lack
the subunits 2 and 5 of this complex and in the
Saccharomyces mitochondrial genome none of the
seven subunits is detectable [ 13, 21 ]. In P. salina
at least subunit 5 of the NADH-dehydrogenase
complex is missing. Whether other subunits are
present and whether this feature could be of tax-
onomical importance should be the subject of
further studies.
So far, taxonomy of algae has been mainly
based on chloroplast pigmentation. It was there-
fore - according to the endosymiont theory - a
taxonomy of the respective former endo-
symbionts. However, this kind of taxonomy
needs to be completed by the respective phylo-
genetic position of the hosts that gave rise to the
modem forms of algae. In case of the cryp-
tomonads several suggestions have been made
concerning the former host. Sequence data of 28S
rRNA [ 34 ] have led to a grouping of crytomonads
together with red algae. In this case, the host as
well as the symbiont are of rhodophytic origin.
This suggestion might be plausible because many
species of red algae are known to be parasites of
other red algae [20]. Considering the fact that
endosymbiosis often is an improved form of para-
sitism in which both partners survive [41 ], crypto-
monads may indeed be descendants of such a
kind of association. However, the mitochondrial
genome of Griffithsia, the only m t D N A of a red
alga described so far, consists of a heterogeneous
population of circular molecules of 40 kb (elec-
tron microscopic analysis) together with linear
molecules of up to 350 kb [31 ]. These data differ

from those we have found for P. salina, where no
heterogeneity in molecule size could be observed.
Moreover, recent forms of flagellated red algae
are not known.
Roberts etal. [35] postulated the host to be
related to the oxymonads or the trichomonads
(Flagellata) according to characteristics displayed
by the flagellar apparatus. Lee and Kugrens [30]
described a suctorian ciliate, Leucocryptos, as an
ancient, still chloroplast-less member of the cryp-
tomonads, which might be similar to the former
host.
To date only few algal mitochondrial genomes
have been analysed. The m t D N A of Euglena con-
sists of circular molecules of 60 kb [15]. The
green alga Chlorella has a circular mitochondrial
genome of about 80 kb [3]. However, m t D N A of
Chlamydomonas reinhardtii (Volvocales) is much
smaller (15 kb [36]) and linear. Similar results
were obtained for another species of the Vovo-
cales, Pandorina morum, which was shown to
possess a linear mitochondrial genome of about
20 kb [33]. For the chromophyte Olisthodiscus
luteus a mitochondrial genome of 50 kb with cir-
cular shape has been demonstrated [ 1 ]. The data
estimated for P. salina m t D N A match well with
those found for other algae, with the exception of
the Volvocales. The Volvocales are supposed to
be an ancient group of green algae, which divided
from other forms early in evolution [48].
On the other hand, mitochondrial genomes of
unicellular eukaryotes such as fungi show such a
wide range of diversities that differences or simi-
larities in molecular size should not be taken as a
taxonomically useful feature. Further information
on gene maps or r R N A sequences from other
unicellular eukaryotes will be necessary to deter-
mine phylogenetic relationships of the former host
on a molecular level.
Acknowledgements
We thank Dr A. Brennicke, Dr W. Schuster and
Dr R.G. Herrmann for kindly providing gene
probes from Oenothera and spinach. We wish to
thank Mrs B. Meltzer and Mrs K. Ronai for
599
technical assistance. The work was supported by
Deutsche Forschungsgemeinschaft (Schwer-
punktprogramm: Endocytosymbiose, grant to
Prof. P. Sitte).
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