Nuclear morphology during the S phase.
In order to evaluate at the ultrastructural level the chromatin arrangement during the S phase of the cell cycle, the detection of Bromodeoxyuridine (BrdU) by immunogold has been performed in synchronized 3T3 fibroblasts, regenerating liver, and Friend Leukemia Cells (FLC). After a 5-minute BrdU pulse, this label is detected in 10-nm-wide fibers, organized as lacework and assumed to be replication units. In the early part of the S phase, DNA replication units are localized exclusively in the dispersed chromatin domains far from the nuclear envelope. In the middle S, replication occurs at the border between condensed and dispersed chromatin and, finally, in late S, it mainly occurs in perinuclear heterochromatin regions. After replication, the 10-nm fibers can condense in heterochromatin without translocation. Chromatin is highly dispersed in early S and computer image analysis shows an increase in condensed chromatin areas ranging from 13 to 18% at the end of the S phase with a temporal and morphological pattern of distribution characteristic for each cell type. Scanning transmission electron microscopy demonstrates a regular and repetitive structure of dispersed chromatin, represented by a ring-like arrangement of the 10-nm fibers; assuming the same spatial distribution, gold particles that identify incorporated BrdU confirm this organization. By evaluating the organization and the distribution of DNA replication units during S phase, the results suggest that DNA replication occurs at a nucleosomal-like fiber level and that replicating enzymes machinery moves over a fixed template.
Mazzotti
G
G
Istituto Anatomia Umana Normale, Università di Bologna, Italy.
Gobbi
P
P
Manzoli
L
L
Falconi
M
M
D016475:3T3 Cells; D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D002467:Cell Nucleus / Q000648:ultrastructure*; D002843:Chromatin / Q000648:ultrastructure; D004261:DNA Replication; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D004915:Leukemia, Erythroblastic, Acute; D008099:Liver / Q000166:cytology / Q000648:ultrastructure; D008115:Liver Regeneration; D008297:Male; D051379:Mice; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D051381:Rats; D017207:Rats, Sprague-Dawley; D016196:S Phase; D014407:Tumor Cells, Cultured
D002843:Chromatin; D001973:Bromodeoxyuridine