Journal of Cell Science 105, 1159-1164 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
1159
Detection of DNA in the nucleoids of chloroplasts and mitochondria in
Euglena gracilis by immunoelectron microscopy
Yasuko Hayashi-Isimaru, Katsumi Ueda* and Mitsuko Nonaka
Biological Laboratory, Nara Women’s University, 630 Nara, Japan
*Author for correspondence
SUMMARY
DNA in the nucleoids of chloroplasts and of mitochondria in Euglena gracilis was detected with anti-DNA
antibodies by immunoelectron microscopy. After treatment with the antibodies, DNA in these organelles combined with gold particles that had been coated with antiIgM antibodies such that it was possible to trace the
outlines of the nucleoids. Nucleoids in chloroplasts
appeared to be composed of twisted threads 50-70 nm
in diameter. The twisted threads were entangled to form
thicker nodes of 100-200 nm diameter. Most nucleoids
in mitochondria were spherical or ovoid, 70-130 nm in
diameter. Nucleoids both in chloroplasts and in mitochondria contained cores with which DNA threads were
in tight contact. The structure of the nucleoids was very
different from those previously observed by conventional electron microscopy.
INTRODUCTION
related fixatives have often been used for the visualization
of nucleoids or ‘DNA fibrils’. In most published reports,
nucleoids appear as electron-transparent regions containing
fine fibrils (Nass and Nass, 1963a,b; Nass et al., 1965;
Yokomura, 1967a,b). In most cases, small numbers of
‘DNA fibrils’ are found in relatively large electron-transparent regions. It has not yet been determined whether
entire transparent regions, without clear boundaries, actually correspond to the nucleoids. Moreover, no evidence
has been presented from which we can infer that only the
fibrous regions correspond to the nucleoids in both chloroplasts and mitochondria. That is, even the outer boundaries
of the nucleoids in chloroplasts and mitochondria have yet
to be determined. Accordingly, detailed morphological
studies on organellar nucleoids are necessary using methods other than conventional electron microscopy.
Immunoelectron microscopy provides reliable information about the distribution pattern of specific antigens at
the ultrastructural level. Using immunogold electron
microscopy, we have visualized the outer boundaries of the
nucleoids and have detected the possible distribution of
DNA in the nucleoids of chloroplasts and mitochondria in
Euglena gracilis as described below.
Chloroplasts and mitochondria contain DNA which appears
to be combined with proteins to form nucleoids in the
organelles (Kuroiwa, 1982; Nemoto et al., 1989). In general, the DNA content of these organelles is far lower than
that of nuclei. Thus, it was previously difficult to detect the
DNA in situ and to visualize the shapes and distribution
patterns of the nucleoids in the organelles.
Use of the dye DAPI for detection of nucleoids by fluorescence microscopy has increased the information that is
available about the shape and the distribution pattern of
nucleoids. Coleman (1978) was the first to succeed in visualizing the nucleoids in chloroplasts, and later she classified the chloroplast nucleoids in various eucaryotic algae
into 6 groups (Coleman, 1985). Kuroiwa et al. (1981) classified the nucleoids in various plants into 5 groups. Changes
in the number, size, and distribution pattern of chloroplast
nucleoids during the life cycle or development of several
algae have been investigated in some detail (Nakamura et
al., 1986; Hatano and Ueda, 1987). Relatively little has been
reported about the behavior of mitochondrial nucleoids
during the cell cycle except in Physarum (Kuroiwa, 1982),
Saccharomyces (Miyakawa et al., 1984), and Euglena
(Hayashi and Ueda, 1989, 1992).
Studies of chloroplast and mitochondrial nucleoids by
fluorescence microscopy are subject to the limits of the
resolving power of the light microscope. Detailed morphological studies should be done by electron microscopy. By
conventional electron microscopy, Kellenberger’s (1958) or
Key words: chloroplast nucleoid, immunocytochemistry,
immunoelectron microscopy, mitochondrial nucleoid, organellar
DNA
MATERIALS AND METHODS
Cells of Euglena gracilis Z strain were cultured at 25°C in
Cramer-Myers medium (Cramer and Myers, 1952), and the culture was illuminated by fluorescent light with a photon flux density of 150 µmol m −2 s−1 for 12 h per day.
1160 Y. Hayashi-Isimaru, K. Ueda and M. Nonaka
For visualization of nucleoids in the mitochondria and chloroplasts, cells were treated with 5 µg/ml DAPI (4′6-diamidino-2phenylindole; Sigma, St. Louis, MO, USA) and were observed by
fluorescence microscopy as described previously (Hayashi and
Ueda, 1989, 1992).
For immunoelectron microscopy, cells were fixed for 1 h at 4°C
in phosphate buffer (pH 7.4) that contained 3% paraformaldehyde
and 2% glutaraldehyde. After washing with water, they were dehydrated with ethanol and embedded in Lowicryl resin at 0°C. Polymerization of the resin was carried out at −25°C under UV light.
Serial sections, each of 70 nm thickness, were attached on Formvar coated nickel glids. Sections were treated successively with
3% H2O2 for 20 min, 3% bovine serum albumin (BSA) for 30
min and 30 µg/ml anti-DNA antibodies (Boehringer Mannheim
Biochemica, Mannheim, Germany) for 2 h at room temperature.
After washing with Tween-PBS (20 mM PBS plus 0.5% Tween20), sections were treated for 1 h at room temperature with colloidal gold (5 nm in diameter) conjugated anti-mouse IgM antibodies (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD,
USA). The gold particles were conjugated with the antibodies by
the method of De May and Moeremans (1986). Sections were
stained with an aqueous solution of 1% uranyl acetate and with
lead citrate. To examine the specificity of the binding between
DNA and the anti-DNA antibodies, treatment of sections with the
antibodies was omitted but the rest of the procedure for detection
of DNA was performed. For digestion of DNA, sections were
immersed for 1 h at 30°C in a buffered solution (pH 5.6) that contained 0.1% DNase I (Worthington Biochem. Corp, NJ, USA) and
1 mM MgSO4. After washing with water, sections were treated
for detection of DNA with the antibodies.
RESULTS
Nucleoids in the chloroplasts and in the mitochondria, visualized by fluorescence microscopy after staining with DAPI,
are shown in Fig. 1. Nucleoids in chloroplasts appeared as
rods of 0.5-2 µm in length. Their diameters cannot be accurately measured because they are so fine. Nucleoids in the
mitochondria appeared as dots. Most nucleoids emitted fluorescence of similar intensity, but one third of the nucleoids
emitted stronger fluorescence.
Fig. 2A shows part of a nucleus, a chloroplast and a mitochondrion that were treated by the immunocytochemical
procedure for detection of DNA. As was anticipated, prominent deposits of gold particles could be seen on the chromosomes in the nucleus. Similar deposits of gold particles
were also visible at places both in the chloroplast and the
mitochondrion. Treatment of sections with DNase during
the immunocytochemical procedure eliminated depositions
of gold particles in defined groups in nuclei, chloroplasts
(Fig. 2B), and mitochondria (Fig. 2C). In the absence of
treatment with the anti-DNA antibodies during the immunocytochemical procedure, no gold particles were also
deposited in groups in nuclei, chloroplasts, or mitochondria
(Fig. 2D).
Seven sectional profiles of chloroplasts are seen in Fig.
3. In each chloroplast, 5-8 small electron-dense regions of
irregular shape are visible, which are the nucleoids. The
high electron-density results from the deposits of gold particles on the nucleoids. The total area of the 7 chloroplasts
is 27.5 µm2 and that of nucleoids 0.51 µm2. Thus the
Fig. 1. Fluorescence of nucleoids in chloroplasts (ch) and
mitochondria (arrows). n, nucleus. Bar, 10 µm.
nucleoids occupy about 1.9% of the cross-sectional area of
the chloroplasts.
Three serial sections of a portion of two chloroplasts are
shown in Fig. 4 (A, B, C). In each panel, there are 6 to 8
regions in which gold particles are distinctly localized.
Deposits of gold particles are seen in similar regions in the
chloroplasts in all three panels, strongly suggesting that the
particles were not deposited randomly. We can conclude
that DNA was present in the three serial sections and that
the gold particles were deposited as a result of the immunological reaction. The sites of the gold particles in the
specific regions of the chloroplasts in the three panels were
superimposed on one another to give Fig. 5. The nucleoid
regions a, b, and c overlap completely with the respective
corresponding regions in the three panels. Region d shifts
to the left gradually as the sections advance from Fig. 4A
to C. There are three regions designated e in Fig. 4A, and
they shift to combine finally as region e in Fig. 4C. Region
f appears in two sections (Fig. 4A,B), while regions g and
h appear in only one section each. Nucleoids a-h are not
completely included within the three sections, and parts of
them may be contained in the sections adjacent to those
shown in Fig. 4.
Fig. 5 shows that the nucleoids are twisted threads of 5070 nm in diameter. The diameters of these threads or the
nucleoids cannot be accurately measured due to undetermined factors concerning the binding pattern between antigens, antibodies, and gold particles at specific regions, so
that the figures are put as approximations. The left halves
of nucleoids a, b, d, and e in Fig. 5 seem to be twisted spirally, and the central portions seem to be wound more than
twice into thicker nodes of 100-200 nm in thickness. Gold
particles are not distributed randomly in the nucleoids but
are arranged along wavy or irregularly folded lines. These
particle-lines may reflect the arrangement of DNA fibrils in
the nucleoids. Transverse sections of the nucleoids reveal
DNA-free central cores and surrounding DNA areas (Fig.
4, arrows). The cores appeared to be slightly more electrondense than the stroma of the chloroplast. Central cores were
35-50 nm in diameter and the total diameter of the
Detection of DNA in the nucleoids 1161
Fig. 3. Distribution of nucleoids in 7 chloroplasts. Electron-dense
regions with irregular outlines represent the nucleoids. Bar, 1 µm.
Fig. 2. (A) Deposits of gold particles on the chromosomes and on
the nucleoids in a chloroplast (ch) and a mitochondrion (m). Bar,
1 µm. DNase treated nucleus and chloroplast (B) and
mitochondria (C). No defined groups of gold particles are visible.
Bar, 1 µm. (D) Treatment with the anti-DNA antibodies was
omitted. No gold-particles were deposited in groups in organelles.
Bar, 1 µm.
nucleoids, including the DNA areas, was 60-100 nm. The
presence of central cores could also be recognized in
oblique sections (Fig. 4, arrowheads). The central cores
seemed not to be completely covered by DNA.
Some profiles of mitochondria were partially covered
with deposits of gold particles. As in the cases of chloroplasts, deposits appeared in the same regions of mitochon-
dria in 2 or 3 successive sections (Figs 6-9). The regions
corresponding to the nucleoids were generally spherical or
ovoid, 70-130 nm in diameter; the nucleoid in Fig. 7 is 100
nm and the nucleoids in Figs 6 and 8 are 120 nm in diameter. Therefore, the regions corresponding to nucleoids were
seen in two successive sections in Fig. 7, and in three successive sections in Figs 6 and 8. Sometimes, large regions
covered with gold particles were seen (Fig. 9). When sites
of gold particles in two successive sections (Fig. 9B,C)
were superimposed, Fig. 9D was obtained. The nucleoid in
Fig. 9D appears to contain DNA threads, 50-70 nm in diameter, that are twisted spirally. DNA threads surrounded
cores in several profiles (Fig. 9B,C, arrows). Cores of mitochondrial nucleoids could also be seen in nucleoids of average size (Fig. 8, arrow). Nucleoids were slightly more electron-dense than other regions of the mitochondria (Fig.
10A,B).
Regions with groups of particles located side by side or
end to end were sometimes observed (Fig. 11A,B). Dumbbell-shaped nucleoids and paired nucleoids have been
observed by fluorescence microscopy in mitochondria, and
they have been interpreted as nucleoids in the process of
division (Hayashi and Ueda, 1989). The groups of particles
located side by side or end to end may correspond to dividing nucleoids.
DISCUSSION
Several reports have described investigations of the distribution of DNA in nuclei and chromosomes with DNAspecific antibodies (Hansmann and Falk, 1986; Thiry and
Thiry-Blaise, 1989; Martin et al., 1992; Thiry, 1992). The
presence of regions in chloroplasts and mitochondria in
which antibody-conjugated gold particles were precipitated
has been reported as an incidental observation in research
processes on the nuclei. For example, Hansmann and Falk
(1986) reported, in addition to their observations of nuclei
and nucleomorphs, that DNA-containing regions were vis-
1162 Y. Hayashi-Isimaru, K. Ueda and M. Nonaka
Fig. 5. Sites of gold particles in the three successive sections in
Fig. 4 were superimposed to generate the respective profiles (see
text for details). ×45,000.
Fig. 4. Nucleoids in chloroplasts in three successive sections (A
through C). Arrows, cores transversely cut; arrowheads, cores
obliquely cut (see text for details). Bar, 0.2 µm.
ible around the center of chloroplasts and that isolated
DNA-containing regions were visible in the mitochondria
of Cryptomonas. In Thiry’s paper (1992), a photograph
showing the deposition of gold particles on chloroplast and
a similar photograph on the mitochondria of Chlamy domonas are included among photographs concerning the
main topic of the detection of DNA within the nucleolus.
One of the essential procedures for immunocytochemical detection of extremely small structures is the examination of serial sections, which is necessary to confirm that
deposits of gold particles appear at the same positions in
organelles in successive sections. This analysis eliminates
the possibility that non-specific or artifactual deposits will
be misinterpreted as specific deposits. Without an examination of serial sections, deposits of antibody-coated gold
particles do not provide convincing evidence for the location of DNA. Previous studies on the detection of organellar DNA failed to include this procedure.
In the present study, gold particles in each serial section
were examined and compared with those in adjacent sections. The deposits in one section matched those in the adjacent sections. Thus, the possibility that the particles were
deposited artifactually is clearly excluded, and the distribution pattern of antigen DNA in the two kinds of organelles
appears convincing.
The figure constructed from 3 serial sections (Fig. 5)
shows that the nucleoids in chloroplasts are rather evenly
distributed in the inter-thylakoid regions and are constructed with twisted threads of 50-70 nm in diameter much
more twisted than can be recognized by fluorescence
microscopy (Fig. 1). The twisted threads were entangled to
form thicker nodes of 100-200 nm in diameter. The way in
which the threads are entangled in the nodes could not be
clarified. As judged from the reconstructed images,
nucleoids in the chloroplasts appeared to have no standard
shape.
Most nucleoids in the mitochondria were spherical or
ovoid and they were smaller than those in the chloroplasts.
However, large nucleoids (Fig. 9) appeared to be composed
of twisted threads of 50-70 nm in diameter, being fundamentally similar in structure to the nucleoids in the chloroplasts.
Mitochondrial nucleoids have been isolated from the
mitochondria of Physarum (Suzuki et al., 1982). They are
0.25-0.3 µm in diameter and 0.7-1.7 µm in length, and they
are composed of DNA filaments that are packed compactly
into three-dimensional rod-shaped structures. They are
larger than those in Euglena and similar in size to the
nucleoids in the chloroplasts of Euglena. The twisting or
the folding of DNA-containing threads seems to be more
complicated in the nucleoids of the chloroplasts of Euglena
than in the isolated mitochondrial nucleoids of Physarum.
One of the important findings in the present study is the
presence of cores in the nucleoids. These cores were found
in the nucleoids of both chloroplasts and mitochondria.
There are two possibilities about the nature of such core;
they may be real cores with some specific chemical con-
Detection of DNA in the nucleoids 1163
Fig. 6. Two mitochondria, each containing a nucleoid with gold deposits, in three successive sections (A through C). Bar, 0.2 µm.
Fig. 7. Mitochondrial nucleoid with gold deposits that appear in two of four successive sections (A through D). Bar, 0.2 µm.
Fig. 8. A mitochondrial nucleoid that appeared in three of five successive sections (A through E). Arrow, nucleoid core. Bar, 0.2 µm.
Fig. 9. A large mitochondrial nucleoid in three successive sections (A through C). Arrows, nucleoid cores. (D) Gold particles in (B) and
(C) are superimposed. Bar, 0.2 µm.
Fig. 10. (A,B) Nucleoids of mitochondria with higher electron-density than matrix. Bar, 0.2 µm.
Fig. 11. Pairs of nucleoids arranged end to end (A) and side by side (B). Bar, 0.2 µm.
1164 Y. Hayashi-Isimaru, K. Ueda and M. Nonaka
stituents or they may be spaces surrounded by DNA threads.
Since most cores were more electron-dense than matrixsubstances, it seems likely that they were real cores. We
plan to examine whether the cores contain histones, RNA,
or enzymes, such as RNA polymerases.
Around the nucleoid regions, which were specified by
the deposition of gold particles, no special regions could be
recognized that correspond to the electron-transparent zones
of nucleoids seen in organelles fixed by Kellenberger’s or
related fixatives (Nass and Nass, 1963a,b; Yokomura,
1967a,b). Moreover, the distribution of gold particles in the
present work was very different from profiles of ‘DNA fibrils’ seen in organelles by those fixations. ‘DNA fibrils’
appeared in some cases as thick fibrils and in other cases
as fine networks without any fundamental organization by
such fixations. Kellenberger’s fixative may introduce artifacts by leaching out certain substances from the nucleoids
and causing aggregation that results in the formation of
‘DNA fibrils’ and the electron transparent zones around
them.
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
REFERENCES
Coleman, A. W. (1978). Visualization of chloroplast DNA with two
fluorochromes. Exp. Cell Res. 114, 95-100.
Coleman, A. W. (1985). Diversity of DNA configuration among classes of
eukaryote algae. J. Phycol. 21, 1-16.
Cramer, M. and Myers, J. (1952). Growth and photosynthetic
characteristics of Euglena gracilis. Arch. Microbiol. 17, 384-402.
De May, J. and Moeremans, M. (1986). The preparation of colloidal gold
probes and their use as marker in electron microscopy. In Advanced
Techniques in Biological Electron Microscopy, vol. 3 (ed. G. Koehler),
pp. 229-271. Berlin, Heidelberg, New York, Tokyo: Springer.
Hansmann, P. and Falk, H. (1986). Ultrastructural localization of DNA in
two Cryptomonas species by use of a monoclonal DNA antibody. Eur.
Cell Biol. 42, 152-160.
Hatano, K. and Ueda, K. (1987). Changes in number of chloroplastnucleoids in the asexual reproduction cycle of Hydrodictyon reticulatum.
Cell Biol. Int. Rep. 11, 13-18.
Hayashi, Y. and Ueda, K. (1989). The shape of mitochondria and the
number of mitochondrial nucleoids during the cell cycle of Euglena
gracilis. J. Cell Sci. 93, 565-570.
Hayashi, Y. and Ueda, K. (1992). Effects of ethidium bromide and
chloramphenicol on the mitochondrial nucleoids in Euglena gracilis.
Physiologia Pl. 86, 57-62.
Kellenberger, E., Ryter, A. and Sechaud, J. (1958). Electron microscope
study of DNA-containing plasms. II. Vegetative and mature phage DNA
as compared with normal bacterial nucleoids in different physiological
states. J. Biophys. Biochem. Cytol. 4, 671-678.
Kuroiwa, T., Suzuki, T., Ogawa, K. and Kawano, S. (1981). The
chloroplast nucleus: distribution, number, size, and shape, and a model
for the multiplication of the chloroplast genome during chloroplast
development. Plant Cell Physiol. 22, 381-396.
Kuroiwa, T. (1982). Mitochondrial nuclei. Int. Rev. Cytol. 75, 1-59.
Martin, M., Moreno Diaz de la Espina, Jimenez-Garcia, L. F.,
Fernandez-Gomez, M. E. and Medina, F. J. (1992). Further
investigations on the functional role of two nuclear bodies in onion cells.
Protoplasma 167, 175-182.
Miyakawa, I., Aoi, H., Sando, N. and Kuroiwa, T. (1984). Fluorescence
microscopic studies of mitochondrial nucleoids during meiosis and
sporulation in the yeast Saccharomyces cerevisiae. J. Cell Sci. 66, 21-38.
Nakamura, S., Ito, S. and Kuroiwa, T. (1986). Behavior of chloroplast
nucleus during chloroplast development and degeneration in
Chlamydomonas reinhardtii. Plant Cell Physiol. 27, 775-784.
Nass, M. M. K. and Nass, S. (1963a). Intramitochondrial fibers with DNA
characteristics. I. Fixation and electron staining reactions. J. Cell Biol. 19,
593-611.
Nass, M. M. K. and Nass, S. (1963b). Intramitochondrial fibers with DNA
characteristics. II. Enzymatic and other hydrolytic treatments. J. Cell
Biol. 19, 613-629.
Nass, M. M. K., Nass, S. and Afzelius, B. A. (1965). The general
occurrence of mitochondrial DNA. Exp. Cell Res. 37, 516-539.
Nemoto, Y., Nagata, T. and Kuroiwa, T. (1989). Studies on plastid-nuclei
(nucleoids) in Nicotiana tabacum L. II. Disassembly and reassembly of
proplastid-nuclei isolated from cultured cells. Plant Cell Physiol. 30, 445454.
Suzuki, T., Kawano, S. and Kuroiwa, T. (1982). Structure of threedimensionally rod-shaped mitochondrial nucleoids isolated from the
slime mould Physarum polycephalum. J. Cell Sci. 58, 241-161.
Thiry, M. and Thiry-Blaise, L. (1989). In situ hybridization at the electron
microscope level: an improved method for precise localization of
ribosomal DNA and RNA. Eur. J. Cell Biol. 50, 235-243.
Thiry, M. (1992). Ultrastructural detection of DNA within the nucleolus by
sensitive molecular immunocytochemistry. Exp. Cell Res. 200, 135-144.
Yokomura, E. (1967a). An electron microscopic study of DNA-like fibrils
in Chloroplasts. Cytologia 32, 361-377.
Yokomura, E. (1967b). An electron microscopic study of DNA-like fibrils
in plant mitochondria. Cytologia 32, 378-389.
(Received 16 February 1993 - Accepted 14 April 1993)