J. Cell Set. 12, 369-383 (1973)
Printed in Great Britain
369
AN ULTRASTRUCTURAL AND
RADIOAUTOGRAPHIC INVESTIGATION OF
THE NUCLEOLONEMAL COMPONENT OF
PLANT INTERPHASE NUCLEOLI
J.-G. LAFONTAINE AND A. LORD
Laboratoire de Biologic ccllulaire et moldculaire,
Dtparttment de Biologie, Faculty des Sciences,
University Laval, Quebec 10, Canada
SUMMARY
The interphase nucleolus in Allhim porrum meristematic cells is characterized by the
presence of 1-4 dense fibrillar zones of rather complex organization. Each such zone appears to
consist essentially of a convoluted, evacuolated, filamentous structure approximately 1-5 /*m
in diameter. At the ultrastructural level, these structures exhibit an intricate array of lacunar
spaces each of which is surrounded by a dense coating. These lacunae are filled with a loose
fibrillar material and the largest ones sometimes also show a dense central core. In appropriate
preparations, certain of the peripherally located lacunae are found to be continuous with
segments of chromosomes. High-resolution radioautography reveals, moreover, that DNA is
present within both the dense and lighter portions of the nucleolar loops. These observations
add further support to the hypothesis that the convoluted filamentous structures in question
correspond to loops of chromosomal origin and are thus related to the nucleolar organizer.
INTRODUCTION
In. plant cells, the nucleolus is known to consist of 2 ultrastructurally distinct
types of zones. Some of these nucleolar portions mostly contain particles which, as
in animal cells, undoubtedly correspond to pre-ribosomal entities (Liau & Perry,
1969; Narayan & Birnstiel, 1969). The remaining nucleolar regions, in plants, are
predominantly fibrillar in texture and, in many of the species examined so far, exhibit
a coarse skein which is thought to consist of loops of chromosome origin (LaCour,
1966). That this hypothesis is basically correct is suggested by both radioautographic
and cytochemical observations showing that such skein-containing nucleolar zones
are the site of synthesis of rapidly labelled RNA (LaCour & Crawley, 1965) and,
moreover, are affected by digestion with deoxyribonuclease (Lord & Lafontaine,
1969; Chouinard, 1970).
It is unfortunately still not clear, however, from these recent studies with plant
material, where exactly DNA is localized within the fibrillar nucleolar zones or, for
that matter, whether it is limited to these nucleolar portions. In view of the large
body of data which has been accumulating in recent years linking nucleolar DNA
with the transcription of pre-ribosomal RNA (Birnstiel, 1967; Miller & Beatty,
1969; Birnstiel, Chipchase & Speirs, 1971), it is of considerable interest to pinpoint
the distribution of this key substance within the nucleolar mass.
37°
J-G. Lafontaine and A. Lord
In the present ultrastructural study, advantage has been taken of the particular
conspicuousness of the nucleolonemal skein in interphase nuclei of Allium porrum
meristematic cells. Owing to the well delimited boundaries of this skein in leek,
this species seemed particularly well suited for verifying with sufficient precision
whether DNA is limited to the nucleolar loop regions, as suggested by earlier observations (LaCour & Wells, 1967). For that purpose, high-resolution radioautography was
exploited and tritium-labelled thymidine used as a marker.
MATERIAL AND METHODS
The observations presented here were carried out on Allium porrum root meristems fixed
and processed according to methods described in a previous article (Lord & Lafontaine, 1969).
For radioautography, roots were immersed in aerated tap water containing 50 /tCi/ml of
[6-'H]thymidine (specific activity 10 Ci/mmol). The remaining portions of the seedlings were
retained above the surface of the solution by means of a perforated plastic support. Since
preliminary observations under electron microscopy revealed that extended exposure of the
roots to thymidine led to a certain degree of segregation of the nucleolar zones, labelling
periods for the present study were limited to 10—30 min. After labelling, the seedlings were
rinsed in water and the root tips excised and fixed. Blocks were sectioned with a Sorval MT-2
ultramicrotome using a diamond knife and light-gold preparations deposited on collodioncoated slides. The latter were then covered with a uniform layer of Ilford L-4 liquid emulsion
by means of the semiautomatic coating instrument described by Kopriwa (1967). Following
appropriate exposure, usually 1-2 months, these preparations were developed by the gold
latensification-Elon ascorbic acid method (Wisse & Tates, 1968). Staining was carried out
with both uranyl acetate and lead citrate solutions according to standard procedures.
RESULTS
Ultrastructure of the interphase nucleolus
In Allium porrum, the interphase chromosomes appear in the form of slender
convoluted strands some 0-3 /im in diameter which, in view of their highly unravelled
condition and complex arrangement within the nuclear cavity, give the impression
of forming a reticulum (Fig. 1).
As this species is tetraploid, interphase nuclei exhibit from 1 to 4 nucleoli depending
whether they have fused or not. As expected from this fusion process, the single
nucleoli may take a dumbbell appearance and are invariably larger than the multiple
ones. In 0-5-0-7/im preparations stained with toluidine blue, interphase nucleoli
show a quite complex organizational pattern; besides sometimes containing vacuoles
of various sizes, they are, indeed, characterized by the presence of one or several
dense irregular zones immersed in a homogeneous and lighter substance. The
examination of serial sections (Figs. 1—6) discloses that these denser zones extend
throughout the mass of the nucleolus and, in fact, correspond to intricate 3-dimensional skein-like structures.
At the electron-microscope level, interphase nucleoli are likewise seen to consist
essentially of 2 types of zones each exhibiting distinct ultrastructural characteristics.
The peripheral portion of the nucleolus, the surface of the centrally located vacuole,
as well as most of the space not occupied by the skein-like areas, consist predominantly of particles some 15-20 nm in diameter interspersed with fine fibrillar material
Radioautography of intranucleolar DNA
371
(Figs. 7, 8). The remaining portions of the nucleolar mass are fibrillar in texture and,
as already apparent from 0-5 /jm preparations, contain a coarse reticulum. One
striking feature of this reticulum is the presence within its meshes of a more transparent fibrillar material which, in favourable preparations, is observed to be intimately
associated with chromosome strands projecting within the nucleolar mass (Fig. 8).
The distribution of these numerous lacunae is seen to much better advantage in the
present micrographs on account of the presence of opaque particles of the type
reported earlier (Lafontaine, 1958; Hyde, Sankaranarayanan & Birnstiel, 1965;
LaCour & Wells, 1969) and which, in A Ilium por rum, are almost completely restricted
to the compact portion of the fibrillar zones. The increased conspicuousness of the
lacunae in such preparations also brings into sharper focus the fact that they are
grouped into 1, 2, 3 and sometimes 4 topographically distinct elongate structures,
the nucleolar skeins, of quite irregular outline and roughly 1-5 /tm in diameter. The
overall appearance of the skeins, as seen in ultrathin preparations, is too intricate for
their 3-dimensional organizational pattern to be easily perceived. Nevertheless, in
Allium porrum, one gains the impression that 3 or 4 rows of more or less linearly
arranged lacunae are present within each such skein. This particular disposition of
the lacunae is most striking at midprophase when the skein becomes fully extended
across the nucleolar mass (authors' unpublished observations).
Radioautographic observations
Preliminary observations of 0-5-0-7 fim thick preparations processed for radioautography revealed restricted thymidine incorporation within the nucleolar mass
even in interphase nuclei which were otherwise heavily labelled over the chromatin.
It also became evident that, for the short incorporation periods used in the present
study, labelling over the nucleolus remained fairly constant whether or not that over
the chromatin was important.
Under electron microscopy, most of the radioautographic grains are found over
the fibrillar portions of the nucleolus. The examination of a hundred or so interphase
nuclei has shown that these grains are localized with equal frequency over both the
thread-like component of these fibrillar zones and the numerous light lacunar spaces
which are closely associated with it (Figs. 9, 10).
Other grains are also frequently observed over the particulate areas of the nucleolus,
but most of these are restricted to the surface of this organelle (Figs. 9-12). This
finding is perhaps not too unexpected, considering that, in Allium porrum as in a
number of other plants with reticulate interphase nuclei, chromatin strands run very
close to the surface of the nucleolus and, at places, may even be rather intimately
associated with it. In a number of interphase nuclei one thus observes several labelled
chromatin strands at various sites around the nucleolus with silver grains extending
to its surface (Fig. 11). Other nuclei show a much more localized labelling pattern
due to the asynchronous DNA duplication process within the chromatin reticulum
(authors' unpublished observations). In some nuclei, for instance, the nucleolus itself
is scarcely labelled, if at all, and only a few restricted portions of the chromatin
reticulum exhibit any significant number of radioautographic grains. Fig. 12 is
37 2
J-G. Lafontaine and A. Lord
quite representative of many such cases observed during the present study and it
illustrates a cluster of grains next to the nucleolar surface. One notes that, here
again, the labelling extends to the peripheral granular portion of the nucleolus
although, in this plane of sectioning at least, none of the chromatin strands seems
to project within the mass of this organelle.
DISCUSSION
In spite of remarkable progress, in recent years, concerning our understanding of
the role of the nucleolus in the economy of the cell (reviewed in Birnstiel, 1967;
Perry, 1969), certain important aspects of its ultrastructural organization are still
rather poorly documented. Foremost among these unsettled problems is that of the
exact localization within the nucleolar body of the polycistronic stretches of DNA
responsible for transcription of ribosomal RNA precursor molecules. As a result, no
consensus has emerged as to which of the various morphological components of
the nucleolus revealed so far by electron microscopy correspond to the nucleolar
organizer proper.
A number of authors working with both animal (Kalnins, Stich & Bencosme, 1964;
von Gaudecker, 1967; Ghosh, Lettre & Ghosh, 1969) and plant (Godward & Jordan,
1965; LaCour, 1966; Jordan & Godward, 1969; Lord & Lafontaine, 1969) cells have,
in recent years, tentatively identified the nucleolonema or a corresponding convoluted
thread-like structure as the nucleolar organizer. Views as to the ultrastructural
organization and cytochemical characteristics of this coarse, filamentous, nucleolar
entity are, however, still somewhat divergent. It has been reported that, in several
types of animal cells, the nucleolonema stains with the Feulgen procedure and
represents an extended part of the nucleolar chromosomes (Lettre, Siebs & Paweletz,
1966; Ghosh, Lettre & Ghosh, 1969). Somewhat more precise information is at
hand in the case of plant material. According to LaCour's (1966) original observations, the skein characterizing plant cell nucleoli consists predominantly of loops of
chromosomal origin which, in certain species, are approximately 1-5 /«m in width
and may reach considerable length (20-55 /" m ) a t interphase. It was hypothesized by
LaCour that these loops contain the nucleolar genes and are thus analogous to the
classical organizer. The presence of chromosomal loops within the fibrillar portions
of the nucleolus has also been conclusively documented in Spirogyra (Godward &
Jordan, 1965; Jordan & Godward, 1969).
The observations reported in the present contribution indicate that each interphase
nucleolus contains one or more skein-like zones which extend throughout its mass
(Figs. 1-6, 11). It has also been found that these complex zones are constant morphological components of the nucleolus till late prophase, at which time this organelle
begins to disorganize (Lafontaine & Lord, in preparation). In view of their intricate
contours it is generally difficult, in ultrathin preparations, to recognize that these
nucleolar skeins consist mainly of coarse meandering filamentous structures. Such
elongate structures may, however, be most convincingly demonstrated in. detergenttreated interphase nucleoli (LaCour, 1966; Lord & Lafontaine, 1969). Fully extended
Radioautography of intranucleolar DNA
373
skeins, approximately 1-5 fim in diameter, are also quite clearly seen running straight
through the nucleolar mass at midprophase and their continuity with certain chromosomes is then much more easily verified (Lafontaine & Lord, in preparation).
In plants it is thus evident that, as the organizer segments penetrate the nucleolus,
they transform into intricate filamentous structures which course throughout the
fibrillar zones of this organelle. These intranucleolar convoluted filaments or nucleolar
loops undoubtedly correspond to the nucleolonema described earlier by Estable
& Sotelo (1955) as well as by various other workers in both plant and animal
cells.
A most intriguing characteristic of the coarse nucleolar loops is the presence of
numerous lacunar spaces which confer to them an evacuolated appearance when
viewed in Tween-dispersed nucleoli or in o-5-//.m stained sections. Under electron
microscopy, these spaces are seen to consist of fine fibrillar material which is generally
distinctly lighter than the chromosomes. However, the fact that, in appropriate preparations, certain of these lacunae are continuous with perinucleolar segments of
chromosomes (Fig. 8) together with their specific labelling with thymidine (Figs. 9,
10) leave little doubt that they actually contain chromatin. Judging from the disposition of the lacunae within each nucleolar loop, it is reasonable to assume that
loose chromatin is therefore running in a most complex fashion within the fibrillar
zones of this organelle.
If, as now seems rather well established, the nucleolonemal skein is related to the
nucleolar organizer proper, how are we to interpret its complex structure as visualized
under electron microscopy? On the basis of their ultrastructural and cytochemical
studies, LaCour & Wells (1967) originally proposed that each nucleolar loop is
formed of a central core of chromatin disposed in a channel of irregular width and
extending along its entire length. Since then, such cores have also been observed in
Allium cepa and shown to react to treatment with DNase (Chouinard, 1970). Since
some of the lacunar cores observed in the present study are specifically labelled with
thymidine (Figs. 9, 10) there seems to remain little doubt that they do indeed contain
DNA. A number of observations point, however, to the possibility that the structural
organization of the nucleolar loops may be more complex than envisaged so far.
In Allium porrum, for instance, dense cores are apparent only within certain of the
larger lacunae. Our radioautographic data suggest, moreover, that DNA is present
not only within the lacunar spaces but also in the immediately surrounding dense
fibrillar coating (Figs. 9—11). Finally, it is of interest to recall that the emerging
telophase nucleolus consists predominantly of a tight glomerular structure which
resembles the non-lacunar portion of the skeins characterizing these organelles at
interphase and prophase (Lafontaine & Lord, 1969). Lacunae appear only later on,
once the late telophase nucleolus has reached a certain size and its glomerular component relaxed to some extent. Such expansion of the lacunar spaces within the skein
coincides with the onset of uridine incorporation over the growing nucleolus (Lafontaine, 1973). This latter phenomenon is, in some respects, analogous to the situation
which arises during activation of dormant cells; the nucleolus-associated chromatin
invades the nucleolar mass and numerous light lacunae become visible within its
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J-G. Lafontaine and A. Lord
fibrillar portions (Jordan & Chapman, 1971). As these changes take place, nucleolar
RNA synthesis is initiated and extensive paniculate zones develop (Rose, Setterfield
& Fowke, 1972).
It is tempting, from these various observations, to hypothesize that the diffuse
intralacunar material corresponds to derepressed chromatin and, as such, constitutes
the active portion of the nucleolar loops. Developing this idea further, one can envisage
that the coarse thread-like portion of each nucleolar skein acts as some sort of support
for fixing the long organizer DNA stretches into a regular and specific pattern as it
spins out of the nucleolar constriction during mid and late telophase.
This investigation was supported by research grants from the Ministry of Education of
Quebec and the National Research Council of Canada. The authors are also grateful to Mrs
Diane Michaud and Mr Siegfried Gugg for their technical assistance.
REFERENCES
M. (1967). The nucleolus in cell metabolism. A. Rev. PL Physiol. 18, 25-58.
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CHOUINARD, L. A. (1970). Localization of intranucleolar DNA in root meristematic cells of
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ESTABLE, C. & SOTELO, J. R. (1955). The behaviour of the nucleolonema during mitosis. In
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GHOSH, S., LETTRE, R. & GHOSH, I. (1969). On the composition of the nucleolus with special
reference to its filamentous structure. Z. Zellforsch. mikrosk. Anat. 101, 254-265.
GODWARD, M. B. E. &JORDAN, E. G. (1965). Electron microscopy of the nucleolus of Spirogyra
britamtica and Spirogyra ellipsospora. Jl R. microsc. Soc. 84, 347-360.
HYDE, B. B., SANKARANARAYANAN, K. & BIRNSTIEL, M. L. (1965). Observations on fine structure in pea nucleoli in situ and isolated. ,7. Ultrastruct. Res. 12, 652-667.
JORDAN, E. G. & CHAPMAN, J. M. (1971). Ultrastructural changes in the nucleoli of Jerusalem
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JORDAN, E. G. & GODWARD, M. B. E. (1969). Some observations on the nucleolus in Spirogyra.
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KALNINS, V. I., STICH, H. F. & BENCOSME, S. A. (1964). Fine structure of the nucleolar
organizer of salivary gland chromosome of chironomids. J. Ultrastruct. Res. n , 282-291.
KOPRIWA, B. M. (1967). A semiautomatic instrument for die radioautographic coating technique. Jf. Histockem. Cytochem. 14, 923-928.
LACOUR, L. F. (1966). The internal structure of nucleoli. In Chromosomes Today, vol. 1 (ed.
C. D. Darlington & K. P. Lewis), pp. 15—160. Edinburgh and London: Oliver & Boyd.
LACOUR, L. F. & CRAWLEY, J. W. C. (1965). The site of rapidly labelled ribonucleic acid in
nucleoli. Chromosoma 16, 124-132.
LACOUR, L. F. & WELLS, B. (1967). The loops and ultrastructure of the nucleolus of Ipheion
uniflorum. Z. Zellforsch. mikrosk. Anat. 82, 25-45.
LACOUR, L. F. & WELLS, B. (1969). The origin of fibrillar particles sometimes seen in plant
nucleoli. Z. Zellforsch. mikrosk. Anat. 97, 358-368.
LAFONTAINE, J. G. (1958). A particulate component found in nucleoli of Allium cepa and Vicia
faba. y. biophys. biochem. Cytol. 4, 229-230.
LAFONTAINE, J. G. (1973). The Nucleus. In Dynamic Aspects of Plant Ultrastructure, chapter
1 (ed. A. W. Robards). New York and London: McGraw-Hill. (In the Press).
LAFONTAINE, J. G. & LORD, A. (1969). Organization of nuclear structures in mitotic cells. In
Handbook of Molecular Cytology (ed. A. Lima-de-Faria), pp. 381-411. Amsterdam and
London: North-Holland Publishing.
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LETTRE, R., SIEBS, W. & PAWELETZ, N . (1966). Morphological observations of the nucleolus of
cells in tissue culture, with special regard to its composition. Natn. Cancer Inst. Monogr. 23,
107-123.
LIAU, M. C. & PERRY, R. P. (1969). Ribosome precursor particles in nucleoli. J. Cell Biol. 42,
272-283.
LORD, A. & LAFONTAINE, J. G. (1969). The organization of the nucleolus in meristematic
plant cells. A cytochemical study. J. Cell Biol. 40, 633-647.
MILLER, O. L. JR. & BEATTY, B. R. (1969). Extrachromosomal nucleolar genes in amphibian
oocytes. Genetics, Princeton, Suppl. 61, 133-143.
NARAYAN, K. S. & BlRNSTiEL, M. L. (1969). Biochemical and ultrastructural characteristics
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ROSE, R. J., SETTERFIELD, G. & FOWKE, L. C. (1972). Activation of nucleoli in tuber slices and
the function of nucleolar vacuoles. Expl Cell Res. 71, 1-16.
VON GAUDECKER, B. (1967). RNA synthesis in the nucleolus of Chironomus thummi as studied
by high resolution autoradiography. Z. Zellforsch. mikrosk. Anat. 82, 536-557.
WISSE, E. & TATES, A. D. (1968). A gold latensification-Elon ascorbic acid developer for Ilford
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{Received 14 July 1972)
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Figs. 1-6. Phase micrographs of 6 consecutive sections of an interphase nucleus
(Allium porrurri). As in other plant species with reticulate interphase nuclei, the
chromosomes consist of strands of quite constant diameter (0-3 )im) but which are
extremely kinky and, therefore, can be followed over short distances only. One large
nucleolus and 3 smaller ones are visible in these serial sections. Each of these interphase nucleoli is seen to consist of dense skein-like regions immersed in a lessstainable pervading substance. Although these dense zones may appear distinct in a
given section, examination of consecutive preparations reveals that they, in fact,
represent portions of an elaborate convoluted filamentous structure, the nucleolonema, which extends throughout the nucleolar mass. The presence of a series of
lacunae confers to the nucleolonema an evacuolated appearance. Although a number
of chromosome strands project towards the surface of the larger nucleolus and, at places,
even approach its nucleolonema (arrows), continuity between these strands and the
latter structure is difficult to establish. This, as is more clearly realized under electron
microscopy (Fig. 8), appears to be due to the fact that the chromatin strands transform into loose, low-staining, intralacunar material as they enter the nucleolus.
Sections 0-5 ^m thick stained with 1 % toluidine blue and photographed under
phase contrast, x 4500.
Radioautography of intranucleolar DNA
371
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J--G. Lafontaine and A. Lord
Fig. 7. Electron micrograph of portion of an interphase nucleus showing a particularly clear view of the composite organization of the nucleolus. Certain portions of the
nucleolar mass, the granular zones (gz), consist predominantly of particulate material
which occupies the peripheral portion of this organelle but also extends more
centrally and thus permeates the spaces in between the 4 denser skein-like nucleolar
regions. Although these latter zones are generally known to consist of fine fibrillar
material, this is not evident here, due to the presence of opaque particles. Nevertheless,
small roundish portions of these fibrillar nucleolar areas are devoid of such particles
and may be seen to have a fibrillar texture. On closer examination, the impression
is also gained that these light lacunar zones more or less correspond to the meshes
of a reticulum formed by the coarse nucleolonemal filament coursing throughout the
fibrillar portions of the nucleolus. The larger of these lacunar regions contain a denser
core (arrows) of fine fibrillar material which stains identically to the extranucleolar
chromatin strands. Note that chromosome segments approach the nucleolar surface
in the immediate vicinity of each of the skein regions, x 44000.
Fig. 8. Portion of an interphase nucleus. The nucleolonema is much more extended
than in Fig. 7 and courses throughout most of the nucleolar mass. As in Fig. 7, however, this denser thread-like nucleolar component circumscribes a series of lacunar
spaces of various shapes and sizes, some of which contain a core (arrows). The present
figure also clearly shows the striking change in density which the nucleolar chromosomes (vch) undergo as they enter the mass of the nucleolus and transform into diffuse
intralacunar material, gz, granular zone; Iz, light lacunar zone, x40000.
Radioautography of intranudeolar DNA
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J-G. Lafontaine and A. Lord-
Figs. 9, 10. Electron-microscopic radioautographs of portions of interphase nuclei
from roots labelled with [6-3H]thymidine for 20 min. Most of the grains are found
over the chromatin strands and nucleoplasm. A lesser but significant number of silver
grains may, however, also be recognized over the lacunar regions (arrows) of the
nucleoli or the surrounding dense fibrillar portions of these organelles. One of the
lacunar cores in Fig. 10 is also labelled. Note, finally, the presence of a few clusters of
grains over the peripheral granular regions of these nucleoli as well as over different
chromatin strands in their immediate vicinity, x 38000 and 45000, respectively.
Radioautography of intranucleolar DNA
«to
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J-G. Lafontaine and A. Lord
Fig. I I . In this interphase nucleus, most of the labelling appears over the chromatin
strands, the nucleolus showing only a few grains over its periphery. Some of these
grains appear to characterize the denser, non-lacunar portion of the nucleolar skein.
x 32000.
Fig. 12. Except for 1 cluster over the nucleolus, all the labelling in this interphase
nucleus is localized over restricted portions of the chromatin reticulum. One such
region is found at the nucleolar surface close to a skein-like region, x 32000.
Radioautography of intranucleolar DNA
383
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