Xenopus embryos initiate replication at random closely spaced sites until a certain concentration of nuclei is achieved within the embryo, after which fewer, more specific chromosomal sites are utilized as origins. We have examined the relationship between nucleo-cytosolic ratio and origin specification when Chinese hamster ovary (CHO) cell nuclei are introduced into Xenopus egg extracts. At concentrations of intact late-G1-phase nuclei that approximate early Xenopus embryos, the entire genome was duplicated nearly 4 times faster than in culture, accompanied by a de-localization of initiation sites at the dihydrofolate reductase (DHFR) locus. As the concentration of nuclei was increased, the number of initiation sites per nucleus decreased and initiation at the DHFR locus became localized to the physiologically utilized DHFR origin. Origin specification was optimal at nuclear concentrations that approximate the Xenopus mid-blastula transition (MBT). Higher concentrations resulted in an overall inhibition of DNA synthesis. By contrast, with intact early G1-phase nuclei, replication initiated at apparently random sites at all concentrations, despite an identical relationship between nucleo-cytosolic ratio and replicon size. Furthermore, permeabilization of late-G1-phase nuclei, using newly defined conditions that preserve the overall rate of replication, eliminated site-specificity, even at nuclear concentrations optimal for DHFR origin recognition. These data show that both nucleo-cytosolic ratio and nuclear structure play important but independent roles in the regulation of replication origin usage. Nucleo-cytosolic ratio clearly influences the number of replication origins selected. However, titration of cytosolic factors is not sufficient to focus initiation to specific sites. An independent mechanism, effecting changes within G1-phase nuclei, dictates which of many potential initiation sites will function as an origin.

REFERENCES

Benavente
R.
,
Krohne
G.
,
Franke
W. W.
(
1985
).
Cell type-specific expression of nuclear lamina proteins during development of Xenopus laevis.
Cell
41
,
177
190
Bielinsky
A.-K.
,
Gerbi
S.
(
1998
).
Discrete start sites for DNA synthesis in the yeast ARS1 origin.
Science
279
,
95
98
Blumenthal
A. B.
,
Kriegstein
H. J.
,
Hogness
D. S.
(
1974
).
The units of DNA replication in Drosophila melanogaster chromosomes.
Cold Spring Harbor Symp. Quant. Biol
38
,
205
223
Brown
E. H.
,
Iqbal
M. A.
,
Stuart
S.
,
Hatton
K. S.
,
Valinsky
J.
,
Schildkraut
C. L.
(
1987
).
Rate of replication of the murine immunoglobulin heavy-chain locus: evidence that the region is part of a single replicon.
Mol. Cell. Biol
7
,
450
457
Buongiorno
,
Nardelli
M.
,
Micheli
G.
,
Carri
M. T.
,
Marilley
M.
(
1982
).
A relationship between replicon size and supercoiled loop domains in the eukaryotic genome.
Nature
298
,
100
102
Chong
J.
,
Thommes
P.
,
Rowles
A.
,
Mahbubani
H.
,
Blow
J.
(
1997
).
Characterisation of the Xenopus replication licensing system.
Meth. Enzymol
283
,
549
564
Coverley
D.
,
Laskey
R. A.
(
1994
).
Regulation of eukaryotic DNA replication.
Annu. Rev. Biochem
63
,
745
776
Diffley
J. F. X.
(
1996
).
Once and only once upon a time: specifying and regulating origins of DNA replication in eukaryotic cells.
Genes Dev
10
,
2819
2830
Dimitrov
S.
,
Almouzni
G.
,
Dasso
M.
,
Wolffe
A. P.
(
1993
).
Chromatin transitions during early Xenopus embryogenesis: changes in histone H4 acetylation and in linker histone type.
Dev. Biol
160
,
214
27
Dimitrov
S.
,
Wolffe
A. P.
(
1996
).
Remodeling somatic nuclei in Xenopus laevis egg extracts: molecular mechanisms for the selective release of histones H1 and H1degfrom chromatin and the acquisition of transcriptional competence.
EMBO J
15
,
5897
5906
Dutta
A.
,
Bell
S.
(
1997
).
Initiation of DNA replication in eukaryotic cells.
Annu. Rev. Cell Dev. Biol
13
,
293
332
Epner
E.
,
Forrester
W. C.
,
Groudine
M.
(
1988
).
Asynchronous DNA replication within the human beta-globin gene locus.
Proc. Nat. Acad. Sci. USA
85
,
8081
8085
Gerber
J.-K.
,
Gogel
E.
,
Berger
C.
,
Wallisch
M.
,
Muller
F.
,
Grummt
I.
,
Grummt
F.
(
1997
).
Termination of mammalian rDNA replication: polar arrest of replication fork movement by tanscription termination factor TTF-1.
Cell
90
,
559
567
Gilbert
D. M.
,
Miyazawa
H.
,
DePamphilis
M. L.
(
1995
).
Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure.
Mol. Cell Biol
15
,
2942
2954
Gilbert
D. M.
(
1998
).
Replication origins in yeast vs metazoa: separation of the haves and the have nots.
Curr. Opin. Gen. Dev
8
,
194
199
Hamlin
J. L.
,
Mosca
P. J.
,
Levenson
V. V.
(
1994
).
Defining origins of replication in mammalian cells.
Biochim. Biophys. Acta
1198
,
85
111
Hyrien
O.
,
Mechali
M.
(
1993
).
Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos.
EMBO J
12
,
4511
4520
Hyrien
O.
,
Maric
C.
,
Mechali
M.
(
1995
).
Transition in specification of embryonic metazoan DNA replication origins.
Science
270
,
994
997
Kill
I. R.
,
Bridger
J. M.
,
Campbell
K. H.
,
Maldonado
C. G.
,
Hutchison
C. J.
(
1991
).
The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts.
J. Cell Sci
100
,
869
876
Kimelman
D.
,
Kirschner
M.
,
Scherson
T.
(
1987
).
The events of the midblastula transition in Xenopus are regulated by changes in the cell cycle.
Cell
48
,
399
407
Kitsberg
D.
,
Selig
S.
,
Brandeis
M.
,
Simon
I.
,
Keshet
I.
,
Driscoll
D.
,
Nicholls
R.
,
Cedar
H.
(
1993
).
Allele-specific replication timing of imprinted gene regions.
Nature
364
,
459
463
Kobayashi
T.
,
Rein
T.
,
DePamphilis
M.
(
1998
).
Identification of primary initiation sites for DNA replication in the hamster DHFR gene initiation zone.
Mol. Cell. Biol
18
,
3266
3277
Lawlis
S. J.
,
Keezer
S. M.
,
Wu
J.-R.
,
Gilbert
D. M.
(
1996
).
Chromosome architecture can dictate site-specific initiation of DNA replication in Xenopus egg extracts.
J. Cell Biol
135
,
1
12
Leibovichi
M.
,
Nonod
G.
,
Geraudie
J.
,
Bravo
R.
,
Mechali
M.
(
1992
).
Nuclear distribution of PCNA during embryonic development in Xenopus laevis: a reinvestigation of early cell cycles.
J. Cell Sci
102
,
63
69
Leno
G. H.
,
Downes
C. S.
,
Laskey
R. A.
(
1992
).
The nuclear membrane prevents replication of human G2nuclei but not G1nuclei in Xenopus egg extract.
Cell
69
,
151
158
Lu
Z. H.
,
Sittman
D.
,
Romanowski
P.
,
Leno
G.
(
1998
).
Histone H1 reduces the frequency of initiation in Xenopus egg extract by lmiting the assembly of prereplication complexes on sperm chromatin.
Mol. Biol. Cell
9
,
1163
1176
Mahbubani
H. M.
,
Paull
T.
,
Elder
J. K.
,
Blow
J. J.
(
1992
).
DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts.
Nucl. Acids Res
20
,
1457
1462
Marshall
W.
,
Fung
J.
,
Sedat
J.
(
1997
).
Deconstructing the nucleus: global architecture from local interactions.
Curr. Opin. Gen. Dev
7
,
259
263
Micheli
G.
,
Luzzatto
A. R.
,
Carri
M. T.
,
DeCapoa
A.
,
Pelliccia
F.
(
1993
).
Chromosome length and DNA loop size during early embryonic development of Xenopus laevis.
Chromosoma
102
,
478
483
Montag
M.
,
Spring
H.
,
Trendelenburg
M. F.
(
1988
).
Structural analysis of the mitotic cycle in pre-gastrula Xenopus embryos.
Chromosoma
96
,
187
196
Newport
J.
,
Kirschner
M.
(
1982
).
A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage.
Cell
30
,
675
686
Newport
J.
,
Kirschner
M.
(
1982
).
A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription.
Cell
30
,
687
696
O'Keefe
R. T.
,
Henderson
S. C.
,
Spector
D. L.
(
1992
).
Dynamic organization of DNA replication in mammalian cell nuclei—spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences.
J. Cell Biol
116
,
1095
1110
Rowles
A.
,
Chong
J.
,
Brown
L.
,
Howell
M.
,
Evan
G.
,
Blow
J.
(
1996
).
Interaction between the origin recognition complex and the replication licensing system in Xenopus.
Cell
87
,
287
296
Sleeman
A. M.
,
Leno
G. H.
,
Mills
A. D.
,
Fairman
M. P.
,
Laskey
R. A.
(
1992
).
Patterns of DNA replication in Drosophila polytene nuclei replicating in Xenopus egg and oocyte extracts.
J. Cell Sci
101
,
509
515
Stick
R.
,
Hausen
P.
(
1985
).
Changes in the nuclear lamina composition during early development of Xenopus laevis.
Cell
41
,
191
200
Stillman
B.
(
1996
).
Cell cycle control of DNA replication.
Science
274
,
1659
1664
Strehl
S.
,
LaSalle
J.
,
Lalande
M.
(
1997
).
High-resolution analysis of DNA replication domain organization across an R/G-band Boundary.
Mol. Cell. Biol
17
,
6157
6166
Taljanidisz
J.
,
Popowski
J.
,
Sarkar
N.
(
1989
).
Temporal order of gene replication in Chinese hamster ovary cells.
Mol. Cell Biol
9
,
2881
2889
Tenzen
T.
,
Yamagata
T.
,
Fukagawa
T.
,
Sugaya
K.
,
Ando
A.
,
Inoko
H.
,
Gojobori
T.
,
Fujiyama
A.
,
Okumura
K.
,
Ikemura
T.
(
1997
).
Precise switching of DNA replication timing in the GC content transition area in the human major histocompatibility complex.
Mol. Cell. Biol
17
,
4043
4050
Walter
J.
,
Newport
J.
(
1997
).
Regulation of replicon size in Xenopus egg extracts.
Science
275
,
993
995
Wang
S.
,
Dijkwel
P.
,
Hamlin
J.
(
1998
).
Lagging-strand, early-labelling and two-dimensional gel assays suggest multiple potential initiation sites in the Chinese hamster dihydrofolate reductase origin.
Mol. Cell. Biol
18
,
39
50
Wu
J.-R.
,
Gilbert
D. M.
(
1996
).
A distinct G1step required to specify the chinese hamster DHFR replication origin.
Science
271
,
1270
1272
Wu
J.-R.
,
Gilbert
D. M.
(
1997
).
The replication origin decision point is a mitogen independent, 2-aminopurine sensitve, G1-phase step that precedes restriction point control.
Mol. Cell Biol
17
,
4312
4321
Wu
J.-R.
,
Keezer
S.
,
Gilbert
D.
(
1998
).
Transformation abrogates an early G1-phase arrest point required for specification of the Chinese hamster DHFR replication origin.
EMBO J
17
,
1810
1818
This content is only available via PDF.