© 1990 Oxford University Press
Nucleic Acids Research, Vol. 18, No. 3
513
Adenovlrus and minute virus of mice DNAs are localized
at the nuclear periphery
Phillip T.Moen Jr, Elizabeth Fox and John W.Bodnar
Northeastern University, Department of Biology, 360 Huntington Ave., Boston, MA 02115, USA
Received October 16, 1989; Revised and Accepted January 3, 1990
ABSTRACT
The localization of adenovirus 2 (Ad2) and Minute Virus
of Mice (MVM) DNAs was studied in situ in infected
HeLa cell nuclei using fluorescent DNA probes and
confocal microscopy. Ad2 DNA was found in multiple
foci which were localized along the periphery of the
infected cell nuclei. MVM DNA was found in HeLa cell
nucleoli which are associated with the nuclear
envelope, and when co-infected with Ad2 MVM DNA
was compartmentalized to multiple foci which again
were localized at the nuclear periphery. The data are
discussed in terms of a model for the role of
intranuclear compartmentalization in eukaryotic DNA
structure and function.
INTRODUCTION
Eukaryotic chromosome structure and function is complicated
by the enormous quantity of DNA contained in a eukaryotic cell.
Since one meter of DNA is contained in each human cell, it
requires complex packaging mechanisms to allow it to be
replicated and expressed and still be contained within a single
cell nucleus. There is growing evidence that the regulation of
both DNA replication and gene expression in eukaryotic cells
is dependent on coordinated regulation of large DNA segments
or 'domains' which may be from ten thousand base pairs to over
two million base pairs in size (reviewed in 1). Therefore, study
of eukaryotic DNA regulation will require a coordinated study
of the biochemistry, molecular and cell biology, and cytology
of defined DNA domains. The complicated interplay of
mechanisms which allow eukaryotic DNA to function have
prompted van Holde to suggest that 'the entire interphase nucleus
may have to be considered as a functional entity' (2). Therefore,
to understand the molecular control of replication and gene
expression the 3-dimensional (3-D) organization of genes within
the interphase nucleus must be understood.
Studies on the intranuclear 3-D organization of DNA have been
limited to date by the specificity or sensitivity of the probes. The
intranuclear arrangement of Drosophila chromosomes has been
reconstructed to show that these chromosomes remain intact
during interphase and have a specific arrangement along the
nuclear envelope (3,4). However, in these studies the probe was
for total DNA and no particular gene could be identified to
determine if the localization along the nuclear envelope was gene
specific. Histological studies of nucleolar structure have indicated
that these organelles are associated with the nuclear envelope in
a variety of animal and plant cells (reviewed in 5), but these
techniques could not resolve the organization of the DNA within
the nucleoli. Analysis of DNAse sensitive DNA in situ indicated
a localization at the nuclear periphery (6), but again no specific
DNA sequences were identified. In situ mapping of cellular DNA
replication sites has shown discrete localizations on the nuclear
matrix for heterochromatin and euchromatin at different times
during S phase; no 3-D spatial localization was shown (7). Since
the current nonisotopic techniques to localize DNA using in situ
DNA hybridization are of low sensitivity, most studies of
intranuclear localization of DNAs have used reiterated sequences,
pooled DNA clones, or cosmids (reviewed in 8). The localization
of an integrated single copy DNA of Epstein-Barr virus has been
characterized (8); using large ( > 5 kb) DNA probes the EBV
DNA was localized within the nuclear interior with high
resolution, but in this case no study was done of flanking cellular
DNA sequences which could potentially be involved in the final
intranuclear compartmentalization of the integrated viral DNA
sequences.
Studies done during the 1970s on the intranuclear localization
of adenovirus DNA gave ambiguous results. Studies using
electron microscopy (EM) indicated that adenovirus DNA is
replicated within the nucleus in multiple foci termed 'replication
factories' and that tritiated adenoviral DNAs appear to be found
throughout the entire nucleus with no preferential association with
the nuclear envelope (9— 11). However, biochemical studies using
the 'M banding' technique indicated that adenovirus DNA
replication is compartmentalized along the nuclear membrane
(10-12).
We have characterized the intranuclear compartmentalization
of adenovirus 2 (Ad2) and Minute Virus of Mice (MVM) DNAs
and have reassessed the previous studies since newer methods
to study nuclear structure are available. Our prior studies have
characterized the biochemical, biophysical, and spatial
compartmentalization of DNA replication of these viral genomes.
Adenovirus DNA is pooled biochemically during infection such
that Ad2 DNA molecules which have just completed a round of
replication can have up to seven times the probability of
replicating again as other Ad2 DNAs, and initiation of replication
of a displaced single-stranded Ad2 DNA intermediate can occur
at greater than fifty times that of the mature Ad2 DNA (13). Both
Ad2 and MVM DNAs are compartmentalized biophysically in
that they are each single DNA domains stably bound to the
nuclear matrix by sequence specific interactions at their DNA
termini. These interactions are most likely mediated by the
514 Nucleic Acids Research
terminal proteins covalently attached to the 5' ends of the linear
viral DNAs (14). Both Ad2 and MVM DNA are
compartmentalized spatially in that they are found in foci within
infected cell nuclei; MVM DNA is localized in nucleoli of
infected cells (15) while Ad2 is localized in multiple non-nucleolar
'replication factories' (9-11,15,16). The Ad2 replication
factories contain several nucleolar antigens which are
recompartmentalized during Ad2 infection, and when MVM is
coinfected with Ad2 in HeLa cells its DNA and terminal protein
are also recompartmentalized from nucleoli to the replication
factories (15). Therefore, Ad2 infection may induce formation
of pseudonucleoli in which its DNA is replicated.
We have now defined the 3-D compartmentalization of Ad2
and MVM DNAs within infected cell nuclei using nonisotopically
labeled DNA probes detected 3-D in situ using confocal
microscopy (17). The Ad2 replication factories were localized
along the nuclear periphery. MVM DNA was replicated within
the nucleoli of infected cells which we found localized along the
nuclear envelope in agreement with previous histological studies
on nucleoli (5). When MVM was coinfected with Ad2 in HeLa
cells MVM DNA replication was recompartmentalized to
replication factories which were localized 3-dimensionally along
the nuclear periphery. These results are discussed in terms of
a model for coordinated 3-D intranuclear compartmentalization
of eukaryotic DNA domains (1) which can also reconcile the
apparent discrepancies in the previous EM and M banding studies
of adenovirus DNA intranuclear localization.
MATERIALS AND METHODS
HeLa cells were grown in suspension or directly on microscope
slides as previously described (13,15). Cells were infected with
adenovirus 2 (MOI = 10), Minute Virus of Mice (MOI = 20),
or coinfected with both viruses, and harvested 24 hours post
infection as previously described (13,15). Suspension cells were
washed in PBS, and dried on microscope slides before fixing
while cells grown on slides were fixed directly. Detailed methods
for both DNA and protein detection in situ were described in
Walton et al. (15). Briefly, for DNA detection cells were fixed
with a series of paraformaldehyde washes, hybridized with DNAs
that had been nick translated with biotinylated dUTP, and the
biotinylated DNAs detected with Texas Red-conjugated
streptavidin. The DNA probe for MVM DNA was plasmid
pPM1816 (15). It was prepared from plasmid pMM984 (18) by
subcloning the 937-base Pst I fragment of MVM genomic DNA
into plasmid pTZ18U (U.S. Biochem. Corp., Cleveland, OH).
Ad2 DNA was prepared from purified virions. Treatment of the
fixed cells with RNAse A prior to DNA hybridization showed
no difference from non-treated cells (data not shown). For protein
detection cells were fixed in methanol-acetone (1:2), probed with
appropriate antisera, and developed with FITC- or TRITCconjugated anti-Ig sera. Antiserum to the Ad5 DNA binding
protein (DBP) was provided by Drs. Vaughn Kleghon and Daniel
Klessig (Waksman Institute) and was developed with Ml'Cconjugated anti-rabbit Ig serum. Antiserum to the Th antigen was
provided by Drs. Susan Baserga and Joan Steitz (Yale University)
and was developed with FITC-conjugated anti-human Ig serum.
Laser confocal microscopy (reviewed in 17) was done in
collaboration with Dr. David Ward (Yale University), using a
Bio-Rad/Microscience Division (Cambridge, MA) MRC-500
Confocal Imaging System and a Nikon Optiphot microscope
(Nikon Instrument Group, Garden City, NY). Excitation of the
FITC or TRITC fluorophores was with an argon laser at 488
run. Optical sectioning was controlled using the BioRad Z-axis
controller, and was performed at 0.5 n intervals through the cell.
Between 20 and 24 sections were obtained for each specimen.
The resulting images were stored on computer for later processing
and image enhancement, using the imaging software supplied with
the MRC-500 Confocal Imaging System.
RESULTS AND DISCUSSION
The 3-D localization of Adenovirus 2 (Ad2) and Minute Virus
of Mice (MVM) DNAs within HeLa cell nuclei was studied by
detection of viral DNAs in situ with nonisotopically labeled DNA
probes followed by optical sectioning of the labeled nuclei by
confocal microscopy (17). In general cells were grown directly
on microscope slides or grown in suspension cultures and fixed
on microscope slides 24 hours post infection with either Ad2,
MVM, or both viruses (13,15). The fixed cells were hybridized
in situ with DNA probes (specific for MVM DNA (15) or Ad2
virion DNA) which had been nick translated with biotinylated
dUTP. The biotinylated DNAs were then detected using Texas
Red-conjugated streptavidin (15). The 3-D localization of the
labeled DNA could be determined by optically sectioning of the
cells (in 0.5 //. sections) using a laser-excited confocal microscope.
The signals from each section were computer enhanced, and the
sections could be studied individually or superimposed to give
a composite of all DNA signals from a single nucleus.
Intranuclear Localization of Ad2 DNA in HeLa Cells
The intranuclear localization of Ad2 DNA in HeLa cell nuclei
was first determined by this method. Cells grown in suspension
are spherical when growing, and the depth of an individual
nucleus is greater than for a cell grown directly on a microscope
slide. Using HeLa cells grown in suspension facilitated separation
of sections within the nucleus although experiments done with
cells grown on slides gave comparable results (see below).
As shown in Figure 1A the distribution of Ad2 DNA within
a HeLa cell nucleus is in multiple discrete foci or 'replication
factories' which are not nucleoli (9—11,15,16). Figure 1A is a
computer reconstructed composite of all optical sections and
represents the total Ad2 DNA in a single HeLa cell nucleus. From
this view the Ad2 DNA is apparently distributed throughout the
nucleus, but a detailed study of the individual optical sections
indicated that much of the Ad2 DNA was in fact localized along
the nuclear periphery (i.e. the DNA which is apparently in the
center of the nucleus was actually at the top or bottom edge of
the nucleus). Note that at 24 hours post-infection, there are about
1 million Adenovirus DNA copies per infected cell nucleus (13).
Individual sections of the same Ad2-infected HeLa cell (Figures
IB through IG) were examined sequentially to indicate the 3-D
organization of the Ad2 DNA. A schematic representation of each
section within the nucleus is also shown next to each section.
It should be noted that although the sections are shown viewed
from the top, for clarity the schematic diagrams are shown in
perspective from the side to better illustrate the 3-D pattern.
Sections were taken at 0.5 n intervals, but alternate sections (1.0 ji
intervals) are presented in the Figures. The intermediate sections
were consistent with presented sections, and foci present in these
sections account for foci seen in composite nucleus (1A) but not
seen in individual sections (1B-1G). Starting from the bottom
of the nucleus (Figure IB) there is a single replication factory
on the bottom surface of the nucleus. As the microscope was
Nucleic Acids Research 515
Ad2 ONA
B
Ad2 DNA
Ad2 DNA
Ad2 DMA
Ad2 DNA
Ad2 DNA
Aci2 ONA
Fig. 1. Intranuclear localization of Ad2 DNA in HeLa cells. Ad2-infected HeLa cells grown in suspension were fixed on microscope slides 24 hours post infection,
hybridized in situ with biotinylated Ad2 virion DNA, the probe DNA detected with Texas Red-conjugated streptavidin (15), and the 3-D distribution of the Ad2
DNA determined using a laser confocal microscope. A. Composite of all optical sections (taken at 0.5 n intervals; typically 20-24 sections from bottom to top
of nucleus) indicating localization of all Ad2 DNA replication factories. A diagrammatic representation of the Ad2 DNA, shown in perspective from the side is
on the left. For all sections, the photographic field shown and the schematic representations are of the cell nucleus only. B - G . Individual serial optical sections
(1 in intervals) starting from the bottom of the HeLa cell. Diagrammatic representation of each cross-section in perspective is shown on the left of each section.
516 Nucleic Acids Research
A
Fig. 2. Intranuclear localization of Th antigen in Ad2-infected and uninfected HeLa cells. Ad2-infected HeLa cells grown in suspension were fixed on microscope
slides 24 hours post infection, probed in situ with Anti-Nuclear Antiserum (ANA) to the Th antigen (which is associated with nucleolar snRNAs |20|), and the
probe detected with FITC-conjugated anti-human Igs. A. Composite of all optical sections (taken at 0.5 n intervals) indicating the localization of all Th antigen in
Ad2-infected (left) and uninfected (right) HeLa cells. A diagrammatic representation of the Th antigen distribution, shown in perspective from the side is below
each cell. B - F . Individual serial optical sections (1 /»intervals) starting from the bottom of the Ad2-infected (left) and uninfected (right) HeLa cells. Diagrammatic
representation of each cross-section in perspective is shown below each cell.
Nucleic Acids Research
focused upward through the nucleus (Figures IB and 1C) the
Ad2 replication factories were evident along the periphery of the
nucleus. In the next section (Figure IE) replication factories were
seen along both edges of the nucleus but a single inclusion was
also detected in the center of the nucleus. As seen in the following
sections (Figures IF and 1G) and in the diagrammatic
representations this spot represented a single large replication
factory extending from the top nuclear surface (Figure 1G) into
the nuclear interior. Four separate Ad2-infected HeLa cell nuclei
were optically sectioned for the localization of Ad2 DNA in the
same way and all exhibited the same pattern—Ad2 DNA was
localized along the nuclear periphery and any replication factories
which were seen in interior portions of any sections corresponded
to foci which extended inward from the edge of that nucleus (data
not shown).
Additionally, when images were computer-reconstructed to
give stereo pairs for 3-D viewing (data not shown), the replication
factories often appeared to form necklace-like patterns where they
were arranged in strings emanating from a single focus. This
may be seen in the diagram in Figure 1A where the group of
foci on the left side of the nucleus starts at the top with the large
inclusion then follows in a string toward the bottom front of the
diagrammatic nucleus while the group of foci of the right side
of the nucleus form a similar string. The repetition of this pattern
in several nuclei suggests that a single input virion can start DNA
replication at the nuclear envelope (the large inclusion at the top
of Figure 1 A) which can then spread along the nuclear envelope
and into the nuclear interior as the infection proceeds.
Nakayasu and Berezney (7) have found that intranuclear
localization of mammalian cell DNA replication is in foci similar
to the adenovirus replication factories. They also found that the
pattern of replication changes over S phase; early in S there are
many replication sites throughout the entire nucleus (Type I sites)
but the pattern changes late in S to fewer replication foci in
heterochromatin (Type HI sites). The Ad2 replication factories
reported here are very similar to the Type HI sites seen by
Nakayasu and Berezney (7), but some Ad2-infected nuclei have
patterns of replication similar to the Type I sites (data not shown).
Therefore, we are continuing to study the pattern and localization
HeLa
517
of Ad2 replication factories as a function of time post-infection,
multiplicity of infection, and cell cycle state at time of infection
(Ek and Bodnar, unpublished results).
To test whether the peripheral localization of Ad2 DNA was
an artifact of the fixing procedure or DNA aggregation or a
characteristic only of suspension cells we probed for the 3-D
localization of the adenovirus 72 kD DNA binding protein (DBP).
Since the adenovirus DBP has been shown to colocalize with the
viral DNA in replication factories (19), we used this as an
independent means to probe for the localization of replication
factories. HeLa cells were grown directly on microscope slides,
infected with Ad2, fixed 24 hours post infection with
methanol:acetone, and probed in situ using antiserum to Ad DBP
followed by development with FITC-conjugated anti-Ig serum.
3-D studies revealed the localization of the DBP in two cells was
the same as the Ad2 DNA (data not shown); the signal was found
in multiple foci localized along the nuclear periphery. In this case
the foci were found mainly along the top or bottom surface of
the flattened nuclei in cells grown directly on microscope slides.
As a further control we also studied the localization of the Th
nucleolar antigen. The Th Anti-Nuclear Antiserum (ANA)
recognizes nucleolar ribonucleoprotein particles containing the
7-2, 8-2 and Ro small nuclear RNAs (snRNAs) (20). The Th
antigen is recompartmentalized in Ad2-infected HeLa cells to a
diffuse pattern throughout the entire nucleus (15). We studied
the 3-D localization of the Th antigen in Ad2-infected and
uninfected HeLa cells side by side on a single microscope slide
(Figure 2). As expected, the Th antigen was in a diffuse nuclear
pattern in an Ad2-infected cell (Figure 2A-left) and in nucleoli
in an uninfected cell (Figure 2A-right). Optical sections of these
cells were photographed as before and alternate sections shown
in Figure 2B to 2F. The Ad2-infected HeLa nucleus shows a
diffuse pattern throughout the nucleus in each separate section
while the uninfected cell is labeled in nucleoli which are found
only near the top edge of the nucleus (Figure 2E and 2F).
It appears that artifactual compartmentalization during sample
preparation can be discounted due to the combined 3-D
localization data for Ad2 DNA, Ad2 DBP, and Th antigen in
side by side Ad2-infected and uninfected HeLa nuclei. Replication
Ad2-infected HeLa
Fig. 3. Intranuclear localization of MVM DNA in HeLa cells and HeLa cells coinfected with Ad2. MVM-infected (left) or MVM- plus Ad2-infected (right) HeLa
cells were grown and fixed on microscope slides 24 hours post infection, hybridized in situ with biotinylated cloned MVM DNA, and the probe DNA detected
with Texas Red-conjugated streptavidin (15). Magnification is 600 x .
518 Nucleic Acids Research
MVM DNA
MVM ONA
B
MVM DNA
MVM DNA
MVM DNA
MVM ONA
Nucleic Acids Research 519
factories studied by Ad2 DNA localization or Ad2 DBP
localization were the same, while Th antigen prepared by identical
means as the DBP samples had two specific patterns that were
both different from the DBP. Additionally, Th was localized in
nucleoli at the nuclear periphery in uninfected HeLa cells which
is consistent with data from several sources that indicate nucleoli
are associated with the nuclear envelope (5).
Our data on 3-D localization of Ad2 replication factories are
consistent with both previous EM and M banding studies for
intranuclear compartmentalization of adenovirus DNA replication
and suggest a way that the apparently contradictory data can be
reconciled. The EM data indicated that tritiated adenovirus DNA
could be found in sections throughout the nucleus (9-11). We
have seen that Ad2 replication factories were associated with the
nuclear envelope, but often they were large and extended into
the nuclear interior. In EM experiments where silver grains
needed to be exposed to determine the location of tritiated DNA
a replication factory which extended into the nuclear interior could
easily be scored as one which was totally within the nuclear
interior. The M banding data indicated that adenovirus DNA
replication was associated with the nuclear membrane (10-12).
These data are also consistent with our results that Ad2 replication
factories are localized along the nuclear envelope.
Additionally, recent EM studies on the nuclear matrix
architecture of Ad2-infected HeLa cells (21) indicated that Ad2
capsids throughout the nucleus were associated with each other
and the nuclear envelope by filaments. From all these data we
suggest that Ad2 DNA replication sites are associated with the
nuclear envelope but that the replicated DNA can be extended
into the nuclear interior as it is 'reeled through' fixed replication
sites (reviewed in 1).
Intranuclear Localization of MVM DNA in HeLa Cells
MVM DNA replicates in the nucleoli of mouse cells, can replicate
weakly in the nucleoli of HeLa cells, and replicates well in
replication factories in Ad2-infected HeLa cells (15). We studied
the 3-D localization of MVM DNA in HeLa cells and found
results which were similar to the localization of the Th antigen
and of Ad2 DNA.
When HeLa cells on microscope slides were infected with only
MVM, a low level of replication is seen which is localized in
the host nucleoli (Figure 3). When these cells were optically
sectioned as before, the result was similar to the Th antigen
pattern (data not shown) in that the signal was found in spots
which were seen only in the sections adjacent to the nuclear
envelope. In most cases it was observed that the MVM DNA
signal was seen in nucleoli located on the bottom of the nucleus
in cells grown on microscope slides (data not shown). This again
is consistent with pr
When MVM DNA is coinfected with Ad2 in HeLa cells
(Figure 3), MVM DNA replicates much more efficiently, and
MVM replication is recompartmentalized to Ad2 replication
factories (15). We estimate that there are several thousand copies
of MVM replicative form DNA per Adenovirus infected HeLa
cell at 24 hours post infection (McHenry, Fox and Bodnar,
unpublished results). HeLa cells grown on microscope slides were
coinfected with MVM and Ad2, fixed 24 hours post infection,
and MVM DNA was detected in situ as before using biotinylated
cloned MVM DNA as a probe (15). MVM DNA was localized
in multiple foci which appeared to be throughout the nucleus
(Figure 4A). When optical sections were studied in sequence
(Figure 4B to 4F show alternate sections as before), the
localization of MVM DNA along the nuclear periphery was again
evident. Prominent replication factories which appeared in the
center of the nucleus were localized on the bottom edge of the
nucleus (Figure 4B). As the sections were taken up through the
nucleus (Figure 4C to 4E) the localization in a ring around the
edge of the nucleus was again apparent. A large replication
factory which appeared to cross the nucleus was found in the
last section (Figure 4F) along the top edge of the nucleus.
Therefore, the 3-D localization data for MVM DNA in HeLa
cells again indicated association with the nuclear envelope either
in nucleoli or replication factories.
A Model for 3-dimensional Intranuclear Organization of Ad2
and MVM DNA
Due to the enormous quantity of DNA that must be packaged
into a single eukaryotic cell, the DNA intranuclear organization
of that DNA must provide for mechanisms whereby the DNA
is packaged, but appropriate cell type-specific genes as well as
DNA replication origins are accessible for regulatory factors.
Several authors have suggested that eukaryotic DNA is organized
into 'domains' often thousand to two million base pairs and that
these DNA domains are the functional units of eukaryotic
chromatin (reviewed in 1). A consolidated model for DNA
domain organization and function has been previously presented
which can account for cell differentiation and genome evolution
in a eukaryotic genome consisting mainly of repetitive sequences
and 'junk' DNA (1). As shown in Figure 5 cellular DNA may
be packaged into DNA domains which are associated with the
nuclear matrix using long range interactions mediated by tightly
bound proteins. Sequences near these stable sites could then serve
as DNA replication origins and signals for cell type-specific
activation of DNA domains. Additional low affinity binding sites
can extend active DNA domains along the fibrillar internal
nuclear matrix to facilitate cell type-specific gene expression.
Implications for this type of DNA organization in cell
differentiation, development, and evolution have been discussed
in detail previously (1,22).
Characterization of intranuclear compartmentalization of Ad2
and MVM DNAs have indicated that the DNAs of both viruses
are organized in DNA domains and can serve as models to study
DNA domain organization (1,13-15). As diagramed in Figure
5 both Ad2 and MVM DNAs are stably associated with the
nuclear matrix at specific sites near their DNA replication origins
and control sequences for early genes, and these interactions are
most likely mediated by proteins covalently bound to the 5'
termini of both DNAs (14). Ad2 DNA is also bound to the
Fig. 4. Intranuclear localization of MVM DNA in HeLa cells coinfected with Ad2. MVM- plus Ad2-infected HeLa cells were grown and fixed on microscope
slides 24 hours post infection, hybridized in situ with biotinylated cloned MVM DNA, the probe DNA detected with Texas Red-conjugated streptavidin (15), and
the 3-D distribution of the MVM DNA determined using a laser confocal microscope. A. Composite of all optical sections (taken at 0.5 n intervals) indicating localization
of all MVM DNA in replication factories. A diagrammatic representation of the MVM DNA is shown in perspective from the side below the HeLa cell. B - F .
Individual serial optical sections (1 n intervals) starting from the bottom of the HeLa cell. Diagrammatic representation of each cross-section is shown in perspective
below each section.
520 Nucleic Acids Research
Ad2
MVM
Fibrillar internal
nuclear matrix
Peripheral
nuclear
matrix
Condensed
Domain
Cell
Active
Domain
Fig. 5. A model for the intranuclear 3-dimensional organization of Ad2, MVM,
and cellular DNA domains. Both Ad2 and MVM DNAs are localized within the
nucleus in a distribution consistent with a DNA domain organization previously
described in detail for DNA domain organization of mammalian cells (1). The
Ad2 and MVM DNAs (>_,) are bound to the peripheral nuclear matrix by stable
interactions of the DNA in their terminal repetitive sequences (EEEI) with proteins
covalently attached to their 5' termini ( • ) . Ad2 DNA is also associated with
the nuclear matrix at multiple low affinity sites (O). A similar organization for
cellular DNA domains has been previously described (1) where DNA domains
are stably bound to the peripheral nuclear matrix by tightly bound proteins ( • )
and active genes extended along nuclear matrix fibrils at multiple low affinity
sites (O) to bring gene coding sequences Q 1) near the nuclear matrix for cell
type-specific expression.
nuclear matrix by low affinity interactions throughout its entire
length (14). This organization is consistent with the
experimentally determined biochemical compartmentalization of
Ad2 DNA replication (13) as well as the spatial
compartmentalization of both Ad2 and MVM DNAs
(9-12,15,16).
Our 3-D localization of Ad2 and MVM DNAs indicates that
domain organization of both viral and cellular DNAs depends
on specific spatial compartmentalization within mammalian cell
nuclei in relation to the nuclear envelope. We suggest that both
Ad2 and MVM DNAs are stably associated with the nuclear
lamina which comprise the stable nuclear matrix along the nuclear
envelope (reviewed in 1) and that these associations are mediated
by the extremely hydrophobic terminal proteins of the two viral
DNAs (1). This would bind both viral DNA replication origins
stably to the nuclear lamina for recognition and subsequent
'reeling through' of DNAs as they are replicated (1). The viral
DNAs can then be extended into the nuclear interior along the
dynamic fibrillar nuclear matrix to allow gene expression by
dynamic site-specific interactions with the internal nuclear matrix.
This model is consistent with all the studies on
compartmentalization of adenovirus within the nucleus. M
banding experiments indicated that adenovirus DNA replication
sites are associated with the nuclear membrane but tritiated DNAs
can be chased from association with the nuclear membrane
(10-12). EM studies have shown tritiated adenovirus in
replication factories throughout the nucleus (9-11), but our 3-D
experiments indicate that these replication factories are anchored
along the nuclear envelope. Finally, Ad2 capsids are associated
with the fibrillar nuclear matrix extending in from the nuclear
lamina (21).
This type of 3-D organization for cellular DNA domains
(Figure 5) is consistent with a wide variety of data on mammalian
DNA domains (1). Of particular interest is a comparison of Ad2
replication factories and host nucleoli. We have found that Ad2
replication factories contain several nucleolar antigens, and
therefore might be pseudonucleoli in which the Ad2 DNA is
complexed with specific nucleolar factors to allow DNA function
(15). This organization is very similar to that suggested for
nucleoli (5) in which nucleoli are anchored along the nuclear
envelope by a stalk of DNA that then enters a complex of proteins
and DNA. Therefore, we suggest that adenovirus and MVM
DNAs can serve as excellent model systems to understand the
role of 3-D intranuclear compartmentalization in the replication
and expression of nucleolar and other cellular DNA domains.
ACKNOWLEDGEMENTS
We thank Dr. George Pearson for helpful suggestions. We thank
Dr. David Ward and Martin Ferguson for the use of the confocal
microscope and assistance in its use. We thank Drs. Susan
Baserga, Joan Steitz, Vaughn Kleghon, and Daniel Klessig for
providing antisera, and we thank William Fowle for assistance
in photography. This project was supported by the National
Institutes of Health (GM35238).
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