High-resolution detection of newly synthesized DNA by anti-bromodeoxyuridine antibodies identifies specific chromatin domains.
We analyzed the incorporation of bromodeoxyuridine (BrdUrd) into DNA in exponentially growing murine erythroleukemia cells (FLC-745), using fluorescent anti-BrdUrd antibodies with light microscopy and flow cytometry. The fine localization of the DNA replicating sites was investigated at the ultrastructural level by using a second antibody conjugated with colloidal gold. The latter approach, which does not require acidic denaturation of the DNA, enables preservation of good morphology and obtains a better resolution power than that of electron microscopic autoradiography, the percentage of labeled cells obtained with the two techniques being comparable. After short BrdUrd pulses, characteristic distribution of the labeling can be identified in the heterochromatin, in interchromatin domains, or at the boundary between the dispersed and the condensed chromatin. Similar patterns are also observable in the nuclear structures which condense after acid denaturation, suggesting that DNA replication takes place at fixed sites associated with the nuclear matrix.
Istituto di Anatomia Umana Normale, Università di Bologna, Italy.
G
G
Mazzotti
R
R
Rizzoli
A
A
Galanzi
S
S
Papa
M
M
Vitale
M
M
Falconi
L M
LM
Neri
N
N
Zini
N M
NM
Maraldi
D000818:Animals; D000906:Antibodies / Q000032:analysis*; D000918:Antibody Specificity; D001973:Bromodeoxyuridine / Q000276:immunology; D002453:Cell Cycle; D002843:Chromatin / Q000032:analysis*; D004261:DNA Replication; D004273:DNA, Neoplasm / Q000096:biosynthesis*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D008854:Microscopy, Electron; D014407:Tumor Cells, Cultured
D000906:Antibodies; D002843:Chromatin; D004273:DNA, Neoplasm; D001973:Bromodeoxyuridine