REPLICATION SITES AS REVEALED BY DOUBLE-LABEL IMMUNOFLUORESCENCE AGAINST PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA) AND BROMODEOXYURIDINE (BRDU) IN SYNCHRONIZED CHO CELLS AND VINCRISTINE-INDUCED MULTINUCLEATE CELLS
H. Takanari et al., REPLICATION SITES AS REVEALED BY DOUBLE-LABEL IMMUNOFLUORESCENCE AGAINST PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA) AND BROMODEOXYURIDINE (BRDU) IN SYNCHRONIZED CHO CELLS AND VINCRISTINE-INDUCED MULTINUCLEATE CELLS, Biology of the cell, 82(1), 1994, pp. 23-31
Double label immunofluorescence against PCNA and BrdU clearly revealed
several characteristics of DNA replication sites in synchronized CHO
cells. We observed that the distribution of replication sites changed
from early to late S phase, particularly in the nucleolar and perinucl
ear regions, and the amount of PCNA in each replication site markedly
decreased or disappeared with the progression of S phase. Although co-
localization of PCNA and BrdU was usually seen, the intensity of fluor
escence occasionally differed between the labeled PCNA and BrdU, parti
cularly in late S phase. Based on the assumption that such a differenc
e may reflect a different configuration of the chromatin, we propose t
hat the brightly granular fluorescent staining of PCNA or BrdU in earl
y S phase indicates a condensed segment of euchromatin. In the late S
phase nucleus, we clearly observed a chromatin-like structure in the l
ate replicating segments of heterochromatin in anti-PCNA stained mater
ial. In vincristine-induced multinucleate cells, a discrepancy between
PCNA-distribution and BrdU-incorporation in sister nuclei was sometim
es seen. Such observations indirectly support the following two mechan
isms for premature chromosome condensation proposed by others: asynchr
onous initiation of DNA synthesis; and a difference in the rate of DNA
synthesis. In addition, the finding that BrdU was not incorporated in
to sites that had PCNA deposits suggests a third mechanism: the local
disturbance of replication sites. Furthermore, unusual distribution pa
tterns for replication sites suggest that the nucleation process in mu
ltinucleate cells differs from that in normal cells.