The relative three-dimensional co-location of RNA polymerase I (RPI) and DNA was studied using confocal laser scanning microscopy during interphase and all the steps of mitosis in human cancerous cells. For each step of the cell cycle, immunolabeled RPI molecules and DNA specifically stained with chromomycin A3 were simultaneously imaged at high resolution through numerous optical sections. Then, all the data obtained were used to generate transverse sections, anaglyphs and volumic representations, which are all prerequisite approaches to a representative study of the three-dimensional organization of the nucleolus and the mitotic chromosomes. Our results indicated that in the interphasic nuclei, in which DNA is organized as a regular 3-D network, RPI was present within numerous irregular spheres arranged as several twisted necklaces. During metaphase, RPI labeling was segregated into pairs of spheres and typical crescent-shaped structures; both were centrally located within the set of chromosomes. During anaphase and telophase, a typical central and symmetric arrangement of labeled structures was systematically seen among the decondensing chromosomes, arranged as a regular cylinder and as a hollow half-sphere, respectively. This typical 3-D organization of structures containing RPI relative to DNA is another strong example of the non-random organization of the genome during interphase and mitosis.

Babu
K. A.
,
Verma
R. S.
(
1985
).
Structural and functional aspects of Nucleolar Organizer Regions (NORs) of human chromosomes.
Int. Rev. Cytol
94
,
151
176
Brelje
T. C.
,
Wessendorf
M. W.
,
Sorenson
R. L.
(
1993
).
Multicolor laser scanning confocal immunofluorescence microscopy: practical application and limitations.
Meth. Cell Biol
38
,
98
182
Carter
K. C.
,
Bowman
D.
,
Carrington
W.
,
Fogarty
K.
,
McNeil
J. A.
,
Fay
F. S.
,
Lawrence
J. B.
(
1993
).
A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus.
Science
259
,
1330
1335
Conconi
A.
,
Widmer
R. M.
,
Koller
T.
,
Sogo
J. M.
(
1989
).
Two different chromatin structures coexist in ribosomal RNA genes throughout the cell cycle.
Cell
57
,
753
761
Derenzini
M.
,
Viron
A.
,
Puvion
E.
(
1984
).
Structural organization of rat hepatocyte chromatin as visualized in thin frozen sections selectively stained for DNA.
Eur. J. Cell Biol
33
,
148
156
Derenzini
M.
,
Thiry
M.
,
Goessens
G.
(
1990
).
Ultrastructural cytochemistry of the mammalian cell nucleolus.
J. Histochem. Cytochem
38
,
1237
1256
Gas
N.
,
Escande
M. L.
,
Stevens
B. J.
(
1985
).
Immunolocation of the 100KDa nucleolar protein during the mitotic cycle in CHO cells.
Biol. Cell
53
,
209
218
Gautier
T.
,
Robert-Nicoud
M.
,
Guilly
M. N.
,
Hernandez-Verdun
D.
(
1992
).
Relocation of nucleolar proteins around chromosomes at mitosis. A study by confocal laser scanning microscopy.
J. Cell Sci
102
,
729
737
Goessens
G.
(
1984
).
Nucleolar structure.
Int. Rev. Cytol
87
,
107
159
Haaf
T.
,
Reimer
G.
,
Schmid
M.
(
1988
).
Immunocytogenetics: location of transcriptionally active rRNA genes in nucleoli and nucleolus organizer regions by use of human autoantibodies to RNA polymerase I.
Cytogenet. Cell Genet
48
,
35
42
Haaf
T.
,
Hayman
D. L.
,
Schmid
M.
(
1991
).
Quantitative determination of rDNA transcription units in vertebrate cells.
Exp. Cell. Res
193
,
78
86
Haaf
T.
,
Schmid
M.
(
1991
).
Chromosome topology in mammalian interphase nuclei.
Exp. Cell Res
192
,
325
332
Hernandez-Verdun
D.
(
1991
).
The nucleolus today.
J. Cell Sci
99
,
465
471
Hozak
P.
,
Cook
P. T.
,
Schöter
C.
,
Mosgöller
W.
,
Wachtler
F.
(
1994
).
Site of transcription of ribosomal RNA and intranucleolar structure in HeLa cells.
J. Cell Sci
107
,
639
648
Hubert
J.
,
Bourgeois
C. A.
(
1986
).
The nuclear skeleton and the spatial arrangement of chromosomes in the interphase nucleus of vertebrate somatic cells.
Hum. Genet
74
,
1
15
Hulspas
R.
,
Bauman
J. G. J.
(
1992
).
The use of fluorescent in situ hybridization for the analysis of nuclear architecture by confocal microscopy.
Cell Biol. Int. Rep
16
,
739
747
Jackson
D. A.
,
Hassan
A. B.
,
Errington
R. J.
,
Cook
P. R.
(
1993
).
Visualization of focal sites of transcription within human nuclei.
EMBOJ
12
,
1059
1065
Jimenez-Garcia
L. F.
,
Rothblum
L. I.
,
Busch
H.
,
Ochs
R. L.
(
1989
).
Nucleologenesis: use of non-isotopic in situ hybridization and immunocytochemistry to compare the location of rDNA and nucleolar proteins during mitosis.
Biol. Cell
65
,
239
246
Jordan
E. G.
(
1991
).
Interpreting nucleolar structure: where are the transcribing genes?.
J. Cell Sci
98
,
437
442
Keiding
N.
,
Andersen
L.
(
1992
).
Estimation of the size distribution of fibrillar centres in nucleoli- an example of the ‘Swiss Cheese’ problem in stereology.
Biometrics
48
,
449
458
Majlof
L.
,
Forgsgren
P. O.
(
1993
).
Confocal microscopy: important considerations for accurate imaging.
Meth. Cell Biol
38
,
79
97
Manuelidis
L.
,
Borden
J.
(
1988
).
Reproductible compartmentlization of individual chromosome domains in human CNS cells revealed by in situ hybridization and three-dimensional reconstruction.
Chromosoma
96
,
397
410
Manuelidis
L.
,
Chen
T. L.
(
1990
).
A unified model of eukaryotic chromosomes.
Cytometry
11
,
8
25
Matsui
S.
,
Sandberg
A. A.
(
1985
).
Intranuclear compartmentalization of DNA-dependent RNA polymerases: association of RNA polymerase I with nuceolar organizing chromosomes.
Chromosoma
92
,
1
6
Mosgöller
W.
,
Leitch
A. R.
,
Brown
J. K. M.
,
Heslop-Harrison
J. S.
(
1991
).
Chromosome arrangements in human fibroblasts at mitosis.
Hum. Genet
88
,
27
33
Ploton
D.
,
Beorchia
A.
,
Menager
M.
,
Jeannesson
P.
,
Adnet
J. J.
(
1987
).
The three-dimensional ultrastructure of interphasic and metaphasicnucleolar argyrophilic components studied with high-voltage electron microscopy in thick sections.
Biol. Cell
59
,
113
120
Ploton
D.
,
Thiry
M.
,
Menager
M.
,
Lepoint
A.
,
Adnet
J. J.
,
Goessens
G.
(
1987
).
Behaviour of nucleolus during mitosis.
Chromosoma
95
,
95
107
Ploton
D.
,
Gilbert
N.
,
Menager
M.
,
Kaplan
H.
,
Adnet
J. J.
(
1994
).
Three-dimensional co-location of nucleolar argyrophilic components and DNA in cell nuclei by confocal microscopy.
J. Histochem. Cytochem
42
,
137
148
Puvion-Dutilleul
F.
,
Bachellerie
J. P.
,
Puvion-Dutilleul
E.
(
1991
).
Nucleolar organization of HeLa cells as studied by in situ hybridization.
Chromosoma
100
,
395
409
Rigaut
J. P.
,
Vassy
J.
,
Herlin
P.
,
Duigou
F.
,
Masson
E.
,
Briane
D.
,
Foucrier
J.
,
Carvajal-Gonzalez
S.
,
Downs
A. M.
,
Mandard
A. M.
(
1991
).
Three-dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks.
Cytometry
12
,
511
524
Robert-Fortel
H. R.
,
Junera
G.
,
Geraud
G.
,
Hernandez-Verdun
D.
(
1993
).
Three dimensional organization of the ribosomal genes and Ag-NOR proteins during interphase and mitosis in PtK1 cells studied by confocal microscopy.
Chromosoma
102
,
146
157
Roussel
P.
,
Andre
C.
,
Masson
C.
,
Geraud
G.
,
Hernandez-Verdun
D.
(
1993
).
Location of the RNA polymerase I transcription factor hUBF during the cell cycle.
J. Cell Sci
104
,
327
337
Scheer
U.
,
Rose
K. M.
(
1984
).
Location of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunochemistry.
Proc. Nat. Acad. Sci. USA
81
,
1431
1435
Scheer
U.
,
Benavente
R.
(
1990
).
Functional and dynamic aspects of the mammalian nucleolus.
BioEssays
12
,
14
21
Schwarzacher
H. G.
,
Wachtler
F.
(
1993
).
The nucleolus.
Anat. Embryol
188
,
515
536
Spector
D. L.
(
1993
).
Macromolecular domains within the cell nucleus.
Annu. Rev. Cell Biol
9
,
265
315
Thiry
M.
,
Scheer
U.
,
Goessens
G.
(
1991
).
Location of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level.
Electron Microsc. Rev.
4
,
85
110
Thiry
M.
(
1993
).
Immunolocation of RNA on ultra-thin sections incubated with polyadenylate nucleotidyltransferase.
J. Histochem. Cytochem
41
,
657
665
Van Driel
R.
,
Humbel
B.
,
de Jong
L.
(
1991
).
The nucleus: a black box being opened.
J. Cell. Biochem
47
,
311
316
Vourc'h
C.
,
Taruscio
D.
,
Boyle
A. L.
,
Ward
D. C.
(
1993
).
Cell cycle-dependent distribution of telomeres, centromeres, and chromosome-specific subsatellite domains in the interphase nucleus of mouse lymphocytes.
Exp. Cell Res
205
,
142
151
Warner
J. R.
(
1990
).
The nucleolus and ribosome formation.
Curr. Opin. Cell Biol
2
,
521
527
Welter
D. A.
,
Black
D. A.
,
Hodge
L. D.
(
1985
).
Nuclear reformation following metaphase in HeLa S3 cells: Three-dimensional visualization of chromatid rearrangements.
Chromosoma
93
,
57
68
This content is only available via PDF.