Download Supercoils in plant DNA: nucleoid

Supercoils in plant DNA: nucleoid sedimentation studies
L. M. STOILOV 1 , J. S. ZLATANOVA1'*, A. P. VASSILEVA1, M. G. IVANCHENKO',
Ch. P. KRACHMAROV 2 and D. D. GENCHEV 1
1
Institute of Genetics and 2Institute of Molecular Biology, Bulganan Academy of Sciences, 1113 Sofia, Bulgaria
* Author for correspondence
Summary
Plant nuclei have been studied with respect to the
three-dimensional structure of DNA. Nucleoids
derived from nuclei by non-ionic detergent and
high salt treatment were analysed by sedimentation in a series of sucrose gradients containing
increasing amounts of the intercalating agent
ethidium bromide. In addition the nucleoid sedimentation behaviour was investigated following
gamma irradiation. The results show that plant
DNA is supercoiled, as is the DNA from the other
eukaryotes studied, and contains approximately
the same concentration of superhelical turns but
probably relatively fewer DNA superhelical
loops. The plant nuclear populations in all cases
studied give rise to two distinct nucleoid bands.
These have been characterized by electron microscopy and by their DNA and protein content.
The possible origin of the two bands is discussed.
Introduction
Kaufmann et al. 1986). The constrained loop concept
comes from microscopic studies and from investigations on histone-depleted nuclei.
Nuclear structures devoid of the nuclear membrane
and of histones by non-ionic detergent and high salt
(2M-NaCl) treatment, called nucleoids (Cook & Brazell, 1975, 1976; Cook et al. 1976), nucleosome-free
interphase chromosomes (Hancock & Hughes, 1982)
and high salt resistant nuclear structures (Mullenders
et al. 1982), have been studied in different animal
systems. These structures preserve the typical nuclear
morphology and behave in a manner characteristic of
intact circular supercoiled DNA. This refers to the way
the sedimentation behaviour of the nucleoids changes
as a function of the ethidium bromide (EthBr) concentration in sucrose density gradients and to the alterations in the sedimentation rate observed following
introduction of single-strand breaks in the DNA by
gamma irradiation (Cook & Brazell, 1975, 1976; Cook
et al. 1976).
It is well known that the final structural organization of
eukaryotic DNA in the nucleus is mediated by several
levels of folding of the long linear DNA molecule. The
primary folding responsible for a six- to sevenfold
compaction of DNA is the winding of the double helix
around the nucleosome core formed by an octamer of
the nucleosomal histones (two molecules each of H2A,
H2B, H3 and H4) (KlugeZ al. 1980). At a second level
the nucleosome fibre (10 nm) is further folded into
thicker fibres (30 nm) organized as supercoils or superbeads, stabilized by histone HI (Finch & Klug, 1976;
Renz et al. 1977). These two levels of folding are still
not sufficient to bring about the final compactness of
the DNA in the interphase nucleus and more particularly in the metaphase chromosome. It is believed that
the thicker fibres are organized in topologically constrained DNA loops anchored to insoluble nuclear
matrix structures. The matrix is isolated by high salt
extraction of nuclei, followed by nuclease treatment
(Berezney & Coffey, 1974, 1977) and consists of a
characteristic set of proteins, whose complexity
depends on the isolation conditions (for review see
Journal of Cell Science 89, 243-252 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
Key words: DNA loops, nucleoid sedimentation, plant
nuclei, supercoiling.
When nucleoids are sedimented in a series of gradients containing increasing concentrations of the intercalating dye EthBr, their sedimentation rates are
243
altered biphasically, the rate first falls and then increases again. This behaviour is explained (by analogy
with the effect of EthBr on covalent close circular
DNA; Vinograd et al. 1965) by the structural changes
due to EthBr intercalation, which first reduces the
number of negative supercoiled turns, then results in a
structure with no net supercoiling and further creates
positive supercoiling (for review see Mattern, 1984).
Irradiated DNA, on the other hand, behaves as if it no
longer contains superhelical DNA, i.e. it behaves like
nicked DNA (Cook & Brazell, 1975). Both these
features of the nucleoid, its sedimentation behaviour
upon EthBr titration and following gamma irradiation,
have been interpreted as an indication that eukaryotic
DNA is supercoiled. Interestingly, similar observations have been reported on the so-called folded
Dmsophila genomes prepared in 0-9M-NaCl, i.e. on
nuclear structures lacking only histone HI but preserving the rest of the histones (Benyajati & Worcel, 1976).
These observations have also been interpreted as
showing the presence of supercoils in Drosophila DNA
loops.
No similar studies aimed at the elucidation of the
higher order organization of nuclear DNA in plants
have been conducted. Plants share many common
features with the other eukaryotes, but there are also
many peculiarities distinguishing them. For example,
they possess unusually large genomes, highly variable
in size and organization from species to species, a lot of
repetitive DNA, low relative amounts of transcribed
sequences, variable ploidy levels (even within a single
plant or tissue), flexibility and plasticity encountered
nowhere else (for reviews see Flavell, 1980, 1982;
Sorenson, 1984; Walbot & Cullis, 1985). The nucleosomal level of organization of the genetic material
seems to be the same as in other eukaryotes (for reviews
see Nagl, 1982fl; Spiker, 1984, 1985). Nothing, however, is known about the secondary and tertiary levels
of DNA folding and also about the relationship between DNA structure and function. In view of the
complete lack of knowledge concerning these issues we
have undertaken a series of investigations, the first of
which is reported here. Using the nucleoid sedimentation method devised by Cook & Brazell (1975, 1976)
we show that plant DNA is also supercoiled. In
addition, we show that nucleoid sedimentation in
plants gives rise to two distinct nucleoid bands; their
nature remains at present unclear and is the subject of
further studies.
Materials and methods
according to the procedure described by Ivanchenko et al.
(1987) either from whole maize (line M320) embryos germinated for different periods of time or from seedling root
meristems. Germination was performed on moist filter paper
at 28°C in the dark and the embryos were dissected by hand.
Microscopic observation of the nuclear pellet showed the
presence of intact nuclei contaminated with some cell debris
and small amounts of starch grains. In some cases nuclei from
Hordeum vulgare and Glycine max were isolated by the same
procedure.
Nucleoid sedimentation assay
All manipulations were carried out at 4°C. Nucleoids were
obtained according to Cook & Brazell (1975, 1976) with some
modifications (Mullenders et al. 1983). Linear sucrose gradients (30ml, 15 % - 3 0 % sucrose, containing 2 M-NaCl, 1 mMEDTA, 25mM-Tris-HCl, p H 8 0 and variable amounts of
EthBr) wre prepared on 1 ml 65 % sucrose cushions containing 0'4gml~' CsCl and were overlaid with 1-5 ml of lysis
solution (0-5% Triton X-100, 2M-NaCl, lmM-EDTA,
25mM-TrisHCl, pH8-0). About 1X106 isolated nuclei
resuspended in 0-5 ml of water were lysed on the top of the
gradients in the dark at 4°C for 30— 60min. The centrifugation was performed in a Sorvall TV-850 ultra vertical rotor
for 4h at 35 000 rev. min" 1 at 4°C. In some cases a Sorvall
AH-650 swinging bucket rotor was used. The position of the
nucleoid bands was determined under 254 nm u.v. light. The
sedimentation rate of nucleoids was expressed relatively to
the sedimentation behaviour of respective controls as specified in the text. Alternatively, gradients were analysed using a
Uvicord fraction collector and recorder operated at 254 nm
(LKB, Sweden). The position of the bands was measured as
the distance from the centre of the absorbance peak to the
bottom of the tube and expressed as a percentage of the
distance covered by the control nucleoids. Both methods gave
comparable results.
Isolation of the DNA and protein components of the
nucleoids
To analyse the DNA and protein components of the nucleoids, they were collected automatically as described above;
the respective bands from several gradients were pooled and
dialysed overnight in the dark at 4°C against 30—50 volumes
of TE buffer (lOmM-Tris- HC1, pH8-0, 1 mM-EDTA, 2
changes). During the dialysis step against this low ionic
strength buffer the proteins dissociated from the DNA and
formed insoluble aggregates, which could be recovered by
low speed centrifugation. After removing the proteins the
supernatant containing practically all nucleoid DNA was
precipitated with cold ethanol and treated with RNase A
(40j*gmr') and proteinase K (lOOjigmF 1 ) for 1 h at 37°C.
The samples were then subjected to a standard phenol
extraction procedure. Finally the samples were dissolved in
small volumes of TE buffer and stored at —20°C for further
analysis. Total nuclear DNA was obtained from isolated
nuclei by phenol extraction preceded by lysis of the nuclei in
the buffer specified above.
Isolation of nuclei
Because plant cells possess thick walls the direct use of cells in
the nucleoid sedimentation assay was not possible and
isolated nuclei were used instead. Nuclei were obtained
244
L. M. Stoilov et al.
Electrophoresis and restriction enzyme digestion
Native and denaturing agarose gel electrophoresis (0"8 %
agarose) of the purified total nuclear or nucleoid DNA was
performed according to Maniatis et al. (1982): A DNA or its
Pst\ digest were used as molecular mass markers. Restriction
enzyme digestions were performed as described by Maniatis
el al. (1982) or according to the instructions of the manufacturer (Promega Biotech, USA).
Proteins were subjected to polyacrylamide gel electrophoresis in slabs containing sodium dodecyl sulphate (Laemmli,
1970).
Determination of G+C content
G + C content was estimated based on the equation of De Ley
(1967) following determination of /?26o/^28o as specified by
Fredericqe/ al. (1961).
Gamma irradiation
A gamma source (CUBE 4000, USSR) containing ^Co was
used to deliver a 10 rad min~' dose rate over a uniform field to
final doses of up to 1000 rad as specified in the text. For the
analysis of the gamma-irradiated material dry seed embryos
or embryos germinated for 16 h were dissected from the
seeds, placed in the homogenization buffer and irradiated at
4°C before isolation of the nuclei. Alternatively, irradiation
was performed on isolated nuclei suspended in homogenization buffer.
Flow cytometry
The flow cytometric analysis was performed on isolated
nuclear suspensions following the procedure described by
Christov & Yantchev (1985). Nuclei were collected by low
speed centrifugation, transferred into staining solution containing 12-5 figml"1 EthBr, 25^gml~' mitramycin and
7-5 mM-MgCl2 and incubated for 15min at 24°C. The
staining was followed by a brief RNase treatment at room
temperature. Between 103 and 10s nuclei were analysed using
an ICP-11 impulse cytophotometer (Phywe, Gottingen, West
Germany).
germinated for 16 h. Two distinct nucleoid bands were
observed under u.v. illumination; the presence of the
two bands was confirmed upon automated fractionation of the gradients (Fig. 1). The same phenomenon
was observed with nuclei from all other maize tissues
tested (dry embryos, root tip meristems, leaves) and
more importantly with nuclei isolated from Hordeum
vulgare root meristematic cells and Glycine max dry
embryos (data not shown). It has to be pointed out that
the relative amounts of the bands varied with the time
of germination. In the dry embryo material the two
bands were present in almost equal amounts. More or
less the same situation was observed at 16 h of germination, but when nucleoids were obtained from root
meristems the intensity of the upper band was much
less than that of the lower one.
As a control purified DNA, either total DNA or from
the individual nucleoid bands, was run in parallel
tubes. All three DNA samples sedimented as single
peaks at nearly the same positions, the position of the
upper nucleoid band. This could mean that the upper
band represented some pure DNA released in the
course of the isolation and centrifugation procedures.
That this was not the case was evident from all further
data on the protein components of the two bands, the
electron microscope pictures and EthBr titrations (see
below). Thus, the upper material is a bona fide
Electron microscopic examination
Nucleoid samples were layered on 0-lM-sucrose cushions
containing 2M-NaCl and 1 % paraformaldehyde and centrifuged in a microcentrifugation chamber for 5 min at 3500 if on
freshly glow-discharged carbon-coated grids. The specimens
were stained in 1 % phosphotungstic acid in 70 % ethanol and
rinsed in absolute ethanol (Miller & Bakken, 1972). Observations were made with a JEM-100B electron microscope at
80kV.
u
Results
Nucleoid sedimentation behaviour of plant material
During the initial phase of our experiments we optimized some of the conditions of the nucleoid sedimentation assay. Thus, best results were obtained with the
lysis solution specified in Materials and methods and
lysis times between 30 and 60 min. It was also essential
to use freshly isolated nuclei as storage of nuclei led to
some smearing of the bands.
Fig. 1 shows a centrifugation tube following sedimentation of nucleoids formed from nuclei of embryos
Fig. 1. Sedimentation of nucleoids from maize embryos
germinated for 16 h in 15%-30% sucrose gradients.
A. Photograph of the centrifugation tube under u.v. light.
B. 254 nm absorbance curve of the same gradient.
Nucleoids were prepared as described in Materials and
methods. Fractionation was from bottom to top. U, upper;
L, lower nucleoid bands.
Supercoils in plant DNA
245
nucleoid band characteristically present in all plant
sources tested.
It is known that restriction enzymes with recognition
sequences CCCGGG or CCGG (Smal, Hpall, Pstl)
do not cleave if the C residue to the 3' side of the
cutting site is methylated (Mann & Smith, 1977). As
20-25 % of the cytosine residues in most higher plant
genomes are methylated (Bedbrook et al. 1978), these
enzymes are not expected to cleave the nuclear DNA
effectively. However, they readily cleave plant extranuclear DNA, giving rise to characteristic ladders of
fragments (for an example of a Pstl digest of chloroplast DNA see Teemuteeri & Lokki, 1984). This
difference in the response of the nuclear and extranuclear genomes to digestion with the above-mentioned
enzymes was used to check whether the two bands were
both of nuclear origin or whether one could be due to
the large amounts of cytoplasmic DNA present in the
plant cell (see e.g. Flavell, 1982). Indeed, as can be
seen from Fig. 2 there was almost complete failure of
Pstl to cleave DNAs from both the lower and the upper
band and total nuclear DNA. As a control for the
reaction, digestion of intact A DNA gave the known
ladder of restriction fragments (Fig. 2, track 2). The
low degree of cleavage observed for the two bands
shows that both contain mainly nuclear DNA.
1
Electron microscopic appearance of the nucleoids and
flow cytometric characterization of the starting
nuclear suspension
The presence of two nucleoid bands could be due to
some variations in the size, shape and/or the density of
the nucleoids. Electron microscopic examination of the
two bands revealed a typical appearance, an electrondense inner mass out of which numerous long DNA
fibres extended to form a dense network interwoven to
different degrees (Fig. 3). The nucleoids of the two
bands did show some differences in compactness and
size, the upper band nucleoids being somewhat
smaller; statistically valid conclusions can however be
drawn on the basis of more experiments. Some differences could also be seen in the appearance of the DNA
halo (compare Fig. 3A and B).
The possibility exists that the two bands reflect
heterogeneity of the starting nuclear populations with
respect to ploidy levels. That the observed two nucleoid bands were not due to heterogeneity in the
amount of DNA per cell (over that due to different cell
cycle phases) is clear from the observation that two
bands were obtained from root meristematic cells.
These cells formed a homogeneous dividing population
with 2C Gi content as evidenced from their distribution histogram according to the DNA content
(Fig. 4). It is known, however, that even in such
homogeneous populations there are several nuclear
phenotypes differing in size and in the amount and
localization of the heterochromatic regions (Vidal et al.
1984). The possibility that the two bands reflect some
of these differences remains to be elucidated. Our
preliminary attempts to fractionate the total nuclear
suspension into different subtypes by the use of stepwise sucrose gradients have been unsuccessful.
The dry maize embryo is known to contain both 2C
and 4C nuclei; moreover, in any population of seeds
there could be variations among seeds depending on
how the metabolism of any seed was arrested during
development and drying (Bewley & Black, 1978). Our
flow cytometric analysis of dry embryo nuclear populations confirmed literature data in that both ZC and 4C
nuclei were present. The proportion of 4C nuclei was
much higher than that in the meristem (not shown).
Again, no nuclei of higher than AC DNA content were
present. This observation confirms the conclusion
drawn from the experiments with the meristematic
cells that the two nucleoid bands are not due to ploidy
heterogeneity.
Fig. 2. Electrophoretic analysis in 0 8 % agarose gels of
the DNA isolated from the two nucleoid bands. Track 1,
total undigested nuclear DNA; tracks 2-4, PstI digests of
A DNA, DNA from the upper and the lower nucleoid
bands respectively; tracks 5, 6, untreated DNA from the
upper and the lower nucleoid bands. Digestion was
performed with equal DNA and enzyme concentrations.
246
L. M. Stoilov et al.
Effect of EtliBr intercalation and irradiation on the
sedimentation behaviour of the nucleoids
As can be seen from Fig. 5 the intercalating agent
EthBr has a significant effect on the sedimentation
properties of the nucleoids. Only the behaviour of the
lower (major) band is presented; the upper band
behaves in a similar manner. As the concentration of
the intercalating dye increased, the distance travelled
by the nucleoid fell to a minimum and then increased
again, the sedimentation rate approaching that of the
control nucleoids, i.e. there was a characteristic biphasic alteration in the sedimentation rates (Fig. 5,
upper curve). There is however a substantial feature
distinguishing the plant nucleoid behaviour from that
observed in the animal systems and it concerns the
magnitude of the difference between the maximum
sedimentation rate (at very low EthBr concentrations)
and its minimum (around 1-2-5 jug ml" 1 EthBr). For
the animal nucleoids the minimal sedimentation rate is
B
about 0-5 of the maximum (Cook & Brazell, 1975)
whereas in our experiments with the 15%-30% sucrose gradients it never decreased below 0-85-0-87 of
the maximum. In less dense gradients of the same
steepness (10 %—25 %) the minimum sedimentation
rate was 0-82 of the maximum; the use of less steep
gradients (15%-25%) did not lead to a significant
change in the value. The sedimentation rate of the
irradiated material was also relatively high (see below).
Gamma rays are known to induce single-strand
scissions in the DNA and irradiation of superhehcal
DNA leads to abolition of the biphasic response of the
Fig. 3. Electron micrographs of
nucleoids from the upper and lower
nucleoid bands obtained from sucrose
gradients under the conditions described
in Materials and methods. A. Upper
band; B. Lower band. Bar, 1 /.an.
Supercoih in plant DNA
247
a 1-0
c
u
•3
•a 0-9
OS
200
60
DNA content (arbitrary units)
100
Fig. 4. Distribution of maize root meristematic cell nuclei
according to their DNA content (arbitrary units), obtained
by flow cytometry.
10
2-5
5
'f
15
Concentration of ethidium bromide
Fig. 5. Sedimentation behaviour of nucleoids derived from
embryos germinated for 16 h as a function of the EthBr
concentration (;tgml~') in the gradient. The sedimentation
rate is presented as a fraction of the distance covered by
control nucleoids (unirradiated material run at 0 1 / i g m l " '
EthBr). Each point is an average value of at least two
independent experiments. (O
O) nucleoids from
unirradiated material; ( •
• ) nucleoids from nuclei
irradiated with 100 rad.
sedimentation behaviour to increasing EthBr concentrations, i.e. irradiated superhelical DNA behaves as a
relaxed molecule. Indeed, when the embryos were
irradiated the biphasic response was completely lost
(Fig. 5, lower curve) (in this case unirradiated material
was used as a control).
It should be noted that the doses that produce these
effects were very small; a similar effect to that presented in Fig. 5 (100 rad) was obtained even when
50 rad were used. Moreover, the dose-response curve
reflecting the changes in the sedimentation behaviour
248
L. M. Stoilov et al.
500
800
Dose of irradiation (rad)
Fig. 6. Dose response curve (sedimentation behaviour of
dry seed nucleoids in 15 % - 3 0 % sucrose gradients versus
dose of gamma irradiation. The sedimentation rate is
calculated as in Fig. 5. (O
O) lower; ( •
• ) upper
nucleoid band.
as a function of the dose of irradiation flattened off at
200 rad (Fig. 6) while similar curves with mouse thymus cells and human lymphocytes continuously
dropped to at least 1000 rad (Weniger, 1982; L. M.
Stoilov, unpublished results).
The existence of two nucleoid bands in the plant
material was totally inexplicable, bearing in mind the
accepted notion that the nucleoids did not sediment as
separate particles but as one aggregate (Weniger, 1982;
Mattern, 1984). Such a notion was based on the
observation that even a mixture of irradiated and nonirradiated cells gave only one DNA peak (Weniger,
1982). In order to check whether the aggregate concept
was also applicable to plants we performed the following mixing experiment. Control nuclei and nuclei
exposed to 500 rad gamma irradiation were mixed in a
1:1 ratio and applied to the top of a gradient; separate
tubes contained only control and only irradiated material. As predicted from the aggregate concept the
mixed nuclei populations gave only two (not four)
nucleoid bands with intermediate sedimentation rates
and of the usual widths (Table 1). Thus it seems that
the aggregate concept is also valid for plants but mixed
aggregates are only formed between otherwise identical
bands whose DNA differs only in the content of singlestrand breaks. Evidently the upper and the lower bands
represent totally distinct nucleoid entities, so different
from one another that they always sediment as individual bands.
Effect of proteinase and RNase treatments on the
nucleoid integrity
When proteinase K and RNase A treatment was
performed during the lysis of the nuclei on top of the
gradients the sedimentation behaviour of the nucleoid
structures changed significantly. When proteinase K
was present in the lysis solution the nucleoid bands
formed became more diffuse and their sedimentation
m
Table 1. Sedimentation behaviour of nudeoids
derived from irradiated and unirradiated nuclei and
from a mixture of the two
Upper band
Lower band
Control
Irradiated
Mixture
59
100
54
92
56-7
97
L
U
X10 " 3
Nuclei were isolated from dry maize embryos and divided into
two portions. One was subjected to gamma irradiation (500 rad),
the other served as control. The mixed nuclear population
consisted of a 1:1 mixture of the irradiated and control material.
The values represent relative sedimentation rates; the distance
covered by the lower band in the control nuclei was used as a
reference. The widths of the corresponding bands was the same
(comparison made on the recorded u.v. profiles by superimposing
the three charts in such a way that the maxima of the lower bands
coincided).
67.
-
45
rate decreased to the values characteristic of the irradiated material. Even more drastic was the effect of
RNase. This treatment done at relatively high enzyme
concentration led to the complete loss of the nucleoid
bands in the tubes (data not shown). Hence this
treatment totally destroyed the integrity of the nucleoids.
General characteristics of the DNA and protein
derived from the nudeoids
DNA from the two bands was isolated by phenol
extraction and compared by agarose gel electrophoresis. As can be seen from Fig. 2 there were no distinct
differences in the electrophoretic properties of the
DNA derived from the two nucleoid bands and of the
total nuclear DNA, run on native gels. All DNAs were
found in the region of 50 kb. Agarose gel electrophoresis under denaturing conditions also failed to show any
significant differences in electrophoretic behaviour
(not shown). As already mentioned the purified DNAs
from the two bands sedimented with similar rates
through sucrose gradients.
Attempts to find differences in the G + C content of
the DNA of the two bands were also unsuccessful. The
ratios of absorbances E26o/Ez80 were determined and
the G + C content was calculated according to the
equation of De Ley (1967). The values obtained were
45-8% for the upper band DNA, 51-6% for the lower
band DNA and 46-5% for the total nuclear DNA.
Bearing in mind the low accuracy of the method we can
infer that the G + C contents of the two bands do not
show significant differences.
The proteins present in the nucleoid bands were
obtained by dialysis against the low ionic strength TE
buffer (see Materials and methods). Under these
conditions the majority of the nucleoid proteins of
molecular mass 45-67K (K = M r Xl0~ 3 ) dissociated
from the DNA as judged by the fact that the DNA in
Fig. 7. Electrophoretogram on 12-5% polyacrylamide gel
of the proteins associated with DNA in maize nudeoids.
m, Molecular mass markers; L, lower nucleoid band;
U, upper nucleoid band.
the buffer contained only trace amounts of these
proteins. The dissociated protein was analysed in
12-5 % polyacrylamide gels containing sodium dodecyl
suphate (Fig. 7). Molecular mass markers and total
acid-soluble chromatin proteins were run for comparison. As seen in Fig. 7 no proteins with the mobility of
histones were present. Interestingly, the two bands
showed exactly the same protein profiles characterized
by the presence of only a few bands in the region of
45—67K. Proteins of similar molecular masses have
been described in HeLa nudeoids (Cook et al. 1976;
Adolph, 1980; Mullenders et al. 1982).
Discussion
Plant DNA contains constrained supercoiled loops
To study the higher order folding of DNA in the plant
nuclei we have investigated the sedimentation behaviour of plant nudeoids in linear sucrose gradients
containing EthBr. When titrated with increasing concentrations of EthBr the nudeoids show the biphasic
Supercoils in plant DNA
249
changes in sedimentation rate characteristic of all
circular supercoiled DNA. In addition, the biphasic
response is completely lost upon induction of singlestrand breaks in the DNA by gamma irradiation. These
observations should be interpreted as showing that
plant DNA is also organized in constrained loops that
contain negative supercoils.
Another important feature concerns the EthBr concentration at the so-called equivalence point (the point
at which EthBr intercalation removes all the negative
superhelical turns and native closed circular DNA
behaves as nicked DNA). The equivalence point in our
experiments is in the range of 1-2-5 Jig m P 1 , i.e. the
same as that observed for SV40 (Crawford & Waring,
1967; Bauer & Vinograd, 1968; Mayer & Levine,
1972), A (Hinton & Bode, 1975), Eschenchia coli
(Worcel & Burgi, 1972), D. melanogaster (Benyajati &
Worcel, 1976) and different mammalian and chicken
sources (Cook & Brazell, 1975, 1976; Cooketal. 1976).
This shows that the superhelical densities in all these
DNA molecules are the same, approximately one
negative superhelical turn per 200bp (Benyajati &
Worcel, 1976).
The significant differences between animal and plant
nucleoids concern two points: the relatively high
sedimentation rate at the equivalence point and of the
irradiated material, and the low radiation doses at
which the dose—response curve levels off. These features might reflect one and the same characteristic of
the plant material. It is not unreasonable to assume, as
Mullenders et al. (1983) have already done, that only
very large DNA loops determine the sedimentation
behaviour of the nucleoids and that the relative amount
of these large DNA loops in the plant genome is low.
An alternative plausible explanation might be a low
relative content of superhelical DNA loops in the plant
sources. Such a low relative content of superhelical
loops might bear some relation to the relatively low
proportion of transcribed and translated DNA sequences in plants; 0-1-1 % of the total DNA in plants
(Nagl, 19826; Flavell, 1982) versus 5-10% in animals
(Pederson, 1978; Lewin, 1975; Mathis et al. 1980). It
should be noted that the same results would have been
obtained if the isolation procedure nicked most of the
nuclear DNA. Although difficult to test, this possibility cannot be ruled out at present.
From studies on animal cell nucleoids it is known
that the integrity of the structure depends on the
presence of protein and RN A (for example see Cook et
al. 1976). The same is evidently true for plant nucleoids as indicated by the proteinase K and RNase A
treatments.
Plant nuclei give rise to two nucleoid bands
The sedimentation of high salt resistant nuclear structures of plants through sucrose gradients revealed
250
L. M. Stoilov et al.
unexpectedly the presence of two bands. Control
mixing experiments (a 1:1 mixture of unirradiated and
irradiated material run in a single tube) showed the
presence of two bands of intermediate sedimentation
rates, which confirmed the aggregate notion of the
sedimentation behaviour of nucleoids (Weniger, 1982).
In addition, they showed, however, that the two bands
normally observed were so different from one another
that it was not possible to form a single band from
them.
Control experiments were run to check whether both
nucleoid bands were of nuclear origin. The high
methylation level of the C residues in the nuclear DNA
was supposed to preclude its effective cleavage with
particular restriction enzymes while chloroplast and
mitochondrial DNA could be cleaved producing
specific restriction fragment patterns. These experiments confirmed the nuclear origin of both nucleoid
bands.
The morphology of the nucleoids was studied by
electron microscopy. The plant nucleoids possess the
typical appearance of their animal counterparts (e.g.
see fig. 2 of Jackson et al. 1984). The structures in the
two nucleoid bands differed mainly in size, the lower
band containing larger structures.
Attempts were made to find some differences either
in the DNA or in the protein content of the two bands.
Isolated DNA was compared electrophoretically, on
the basis of its G + C content and its sedimentation in
sucrose gradients. In no respect could differences be
detected. The same applied to the protein content of
the two bands; the electrophoretic patterns were indistinguishable, characterized by the presence of only a
few bands in the region of 45—67K. The proteins of
animal cell nucleoids gave very similar electrophoretic
patterns (Cook et al. 1976; Adolph, 1980; Mullenders
et al. 1982). Some of the proteins have been characterized as lamins, the major components of the nuclear
pore complex-lamina. Recently, similar proteins have
been described in isolated plant nuclear matrices
(Ghosh & Dey, 1986). On the basis of the similarities
between the plant and animal nucleoid proteins it
might be suggested that the proteins taking part in
DNA loop formation are highly conserved. Our preliminary data using peptide mapping and immunochemical techniques do confirm a great degree of
conservation of these proteins.
The existence of the two nucleoid bands in plants
could reflect some heterogeneity of the nuclear populations with respect to size, shape and/or density. That
the two bands are not due to different ploidy levels is
evident from the observations that a dividing diploid
population, as represented by the root meristematic
cells, also gives two bands.
The possibility that cell cycle-dependent differences
in nuclear parameters determine the presence of two
bands cannot be excluded at present. HeLa cell nueleoids derived from synchronous Gi, S and M populations did show significant differences in the relative
sedimentation rate, small S phase nueleoids sedimenting nine times faster than the larger mitotic nueleoids
(Warren & Cook, 1978). While this difference is
probably too great to allow separation of S and M
nueleoids within a single standard gradient, such a
separation would be expected for S and Gi nueleoids
whose sedimentation rate differed by only 20-30%.
Nevertheless, nueleoids from unsynchronized populations never exhibited two bands (Cook & Brazell,
1975, 1976; Cook et al. 1976; Warren & Cook, 1978).
The reason for this discrepancy remains unclear; the
aggregate notion of nucleoid sedimentation behaviour
could be a plausible explanation. If the two plant bands
do reflect cell cycle-dependent differences, these
should be highly specific for plants, as they do not allow
the formation of a single aggregate out of the distinct
phase nueleoids.
BENYAJATI, C. & WORCEL, A. (1976). Isolation,
In summary, the nucleoid sedimentation studies
performed in this work lead to the following main
conclusions.
(1) Plant DNA contains supercoiled loops with the
same superhelical density as observed for phage, viral,
bacterial, insect and vertebrate genomes. The relative
amount of superhelical loops in the plant genome seems
to be much lower than in the animal genome, correlating with its much lower transcriptional activity. The
proteins responsible for the constraining of the DNA
loops are very similar between plants and animals.
(2) A characteristic feature of plant nuclear populations is that they form two distinct nucleoid bands,
indistinguishable by all criteria used in this study.
Further investigations at the cellular and molecular
levels are required to clarify this point.
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The authors thank Dr K. Christov for performing the flow
cytometric analysis and Dr L. Todorova for supplying the
dry maize seeds. This project has been completed with the
financial support of the Committee for Science at the Council
of Ministers under contract 486.
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(Received 10 August 1987 -Accepted, in revisedfonn,
3 Xovember 1987)