The temporal and spatial progression of DNA replication in interphase nuclei of eukaryotic cells has been investigated. Application of a recently developed technique for the immunofluorescence double staining of cell nuclei labelled first with iododeoxyuridine (IdUrd) and subsequently with chlorodeoxyuridine (CldUrd) allows the visualization of two replication patterns in the same nucleus originating from two different periods of the S-phase. We have analysed changes in the three-dimensional replication patterns during the S-phase. To record dual colour three-dimensional images of doubly stained nuclei, a confocal microscope is used. This CSLM is equipped with a specific laser/filter combination to collect both fluorescence signals (FITC and Texas Red) in a single scan, thus precluding pixel shift between the images. A method for the quantitative evaluation of the degree of overlap between DNA regions replicated in two different periods of the S-phase is applied. The results confirm the generally accepted theory that DNA is replicated coordinately in a specific temporal order during the S-phase. The replication time of a DNA domain (i.e. the time between initiation and termination of DNA replication within a domain) at the very beginning of the S-phase was known to be one hour (Nakamura et al., 1986). Our observations show that in the rest of the S-phase, the replication time of a DNA region is also about one hour. We conclude that replicon clusters located in the same region are replicated in the same relatively short period of time. After this period there is no unreplicated DNA left in this region.

Aten
J. A.
,
Bakker
P. J. M.
,
Stap
J.
,
Boschman
G.A.
,
Veenhof
C. H. N.
(
1992
).
DNA double labelling with IdUrd and CldUrd for spatial and temporal analysis of cell proliferation and DNA replication.
Histochem. J
24
,
251
259
Bakker
P. J. M.
,
Stap
J.
,
Tukker
C. J.
,
Oven
C. H.
,
van, Veenhof
C. H. N.
,
Aten
J.
(
1991
).
An indirect immunofluorescence double staining procedure for the simultaneous flow cytometric measurement of iodo-and chlorodeoxyuridine incorporated into DNA.
Cytometry
12
,
366
372
Banfalvi
G.
,
Tanke
H.
,
Raap
A. K.
,
Slats
J.
,
Van der Ploeg
M.
(
1990
).
Early replication signals in nuclei of Chinese hamster ovary cells.
Histochemistry
94
,
435
440
Berezney
R.
,
Coffey
D. S.
(
1975
).
Nuclear protein matrix: association with newly synthesized DNA.
Science
189
,
291
293
Brakenhoff
G. J.
,
Van der Voort
H. T. M.
,
Van Spronsen
E. A.
,
Linnemans
W. A. M.
,
Nanninga
N.
(
1985
).
Three dimensional chromatin distribution in neuroblast nuclei shown by confocal scanning laser microscopy.
Nature
317
,
748
749
Brakenhoff
G.J.
,
Van der Voort.
H. T. M.
,
Van Spronsen
E. A.
,
Nanninga
N.
(
1989
).
Three dimensional imaging in fluorescence by confocal scanning microscopy.
J. Microsc
153
,
151
159
Cook
P. R.
(
1991
).
The nucleoskeleton and the topology of replication.
Cell
66
,
627
635
Dean
P.
,
Mascio
L.
,
Ow
D.
,
Sudar
D.
,
Mullikin
J.
(
1990
).
Proposed standard for image cytometry data files.
Cytometry
11
,
561
569
Dierendonck
J. H.
,
van
Kayzer, R.
,
van de Velde
C. J. H.
,
Cornelisse
C. J.
(
1989
).
Subdivision of S-phase by analysis of nuclear 5-bromodeoxyuridine staining patterns.
Cytometry
10
,
143
150
Edenberg
H. J.
,
Huberman
J. A.
(
1975
).
Eukaryotic chromosome replication.
Annu. Rev. Genet
9
,
245
284
Fakan
S.
,
Hancock
R.
(
1974
).
Localization of newly-sythesized DNA in a mammalian cell as visualized by high resolution autoradiography.
Exp. Cell Res
83
,
95
102
Fox
M. H.
,
Arndt-Jovin
D. J.
,
Jovin
T. M.
,
Baumann
P. H.
,
Robert-Nicoud
M.
(
1991
).
Spatial and temporal distribution of DNA replication sites localized by immunofluorescence and confocal microscopy in mouse fibroblast.
J. Cell Sci
99
,
247
253
Gratzner
H. G.
(
1982
).
Monoclonal antibody to 5-bromo and 5-iododeoxyuridine: A new reagent for detection of DNA replication.
Science
218
,
474
475
Hand
R.
(
1978
).
Eucaryotic DNA: Organization of the genome for replication.
Cell
15
,
317
325
Housman
D.
,
Huberman
J. A.
(
1975
).
Changes in rate of DNA replication fork movement during S-phase in mammalian cells.
J. Mol. Biol
94
,
173
181
Huberman
J. A.
,
Riggs
A. D.
(
1968
).
On the mechanism of DNA replication in mammalian chromosomes.
J. Mol. Biol
32
,
327
341
Huberman
J. A.
,
Tsai
A.
,
Deich
R. A.
(
1973
).
DNA replication sites within nuclei of mammalian cells.
Nature
241
,
32
36
Jackson
D. A.
(
1990
).
The organization of replication centres in higher eukaryotes.
BioEssays
12
,
87
89
Laskey
R. A.
,
Fairman
M. P.
,
Blow
J. J.
(
1989
).
S-phase of the cell cycle.
Science
246
,
609
621
Mazzotti
G.
,
Rizzoli
R.
,
Galanzi
A.
,
Papa
S.
,
Vitale
M.
,
Falconi
M.
,
Neri
L. M.
,
Zini
N.
,
Maraldi
N. M.
(
1990
).
High-resolution detection of newly sythesized DNA by anti-bromodeoxyuridine antibodies identifies specific chromatin domains.
J. Histochem. Cytochem
38
,
17
22
Mills
A. D.
,
Blow
J. J.
,
White
J. G.
,
Amos
W. B.
,
Wilcock
D.
,
Laskey
R. A.
(
1989
).
Replication occurs at discrete foci spaced throughout nuclei replicating in vitro.
J. Cell Sci
94
,
471
477
Nakamura
H.
,
Morita
T.
,
Sato
C.
(
1986
).
Structural organizations of replicon domains during DNA sythetic phase in the mammalian nucleus.
Exp. Cell Res
165
,
291
297
Nakayasu
H.
,
Berezney
R.
(
1989
).
Mapping replicational sites in the eucaryotic cell nucleus.
J. Cell Biol
108
,
1
11
Padroll
D. M.
,
Vogelstein
B.
,
Coffey
D. S.
(
1980
).
A fixed site of DNA replication in eucaryotic cells.
Cell
19
,
527
536
Smith
H. C.
,
Puvion
E.
,
Buchholtz
L. A.
,
Berezney
R.
(
1984
).
Spatial distribution of DNA loop attachment and replicational sites in the nuclear matrix.
J. Cell Biol
99
,
1886
1894
Van Dekken
H.
,
Van Rotterdam
A.
,
Jonker
R. R.
,
Van der Voort
H. T. M.
,
Brakenhoff
G. J.
,
Bauman
J. G. J.
(
1990
).
Spatial topography of a pericentromeric region (1q12) in hemopoietic cells studied by in situ hybridization and confocal microscopy.
Cytometry
11
,
570
578
Villarreal
L. P.
(
1991
).
Relationship of eucaryotic DNA replication to committed gene expression: General theory for gene control.
Microbiol. Rev.
55
,
512
542
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