Nuclear matrix-bound replicational sites detected in situ by 5-bromodeoxyuridine.
The nuclear matrix was prepared in situ from Swiss 3T3 cells, which were synchronized by contact inhibition and serum starvation and pulse-labelled for very short periods of time with 5-bromodeoxyuridine (5-BrdU). For the first time 5-BrdU has been employed to demonstrate the association of newly synthesized DNA with a nucleoskeleton. Immunofluorescence analysis using a monoclonal antibody to 5-BrdU revealed five different intranuclear staining patterns at different stages of the S phase. These patterns were observed also in intact cells and did not change during the matrix preparation steps which involve extraction with 2 M NaCl and DNase I digestion. Such an observation was also confirmed by spatial confocal microscopy studies. The intensity of fluorescence, which was evaluated by cytofluorometry, increased to reach a maximum during mid-S phase and then decreased. Because no significant difference was found in the time to label residual DNA of different 5-BrdU staining patterns, this strongly suggests that a different number of replicons is activated at different stages of the S phase. These results strengthen the hypothesis that eukaryotic DNA replication occurs in close association with an insoluble protein nuclear skeleton, which determines the three-dimensional spatial organization of chromosome duplication.
Neri
L M
LM
Istituto di Anatomia Umana Normale, Universitá di Ferrara, Italy.
Mazzotti
G
G
Capitani
S
S
Maraldi
N M
NM
Cinti
C
C
Baldini
N
N
Rana
R
R
Martelli
A M
AM
D016475:3T3 Cells / Q000378:metabolism / Q000502:physiology; D000818:Animals; D001973:Bromodeoxyuridine; D002467:Cell Nucleus / Q000502:physiology; D016895:Culture Media, Serum-Free; D004247:DNA / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D051379:Mice; D015530:Nuclear Matrix / Q000378:metabolism / Q000502:physiology*; D012093:Replicon / Q000502:physiology*
D016895:Culture Media, Serum-Free; D004247:DNA; D001973:Bromodeoxyuridine