In vitro DNA Synthesis in the Macronuclear Replication Band of Euplotes eurystomus

Isolated macronuclei from the hypotrichous ciliated protozoan Euplotes eurystomus incorporate biotinylated dUTP specifically into the replication band (RB) as detected with immunofluorescence, using rabbit anti-biotin antibodies followed by fluorescein-conjugated goat anti-rabbit IgG. When gold-conj...

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Published inThe Journal of cell biology Vol. 104; no. 5; pp. 1125 - 1132
Main Authors Olins, Donald E., Olins, Ada L.
Format Journal Article
LanguageEnglish
Published New York, NY Rockefeller University Press 01.05.1987
The Rockefeller University Press
Subjects
Animals
Biological and medical sciences
Cell Nucleus - metabolism
Cell Nucleus - ultrastructure
Cells
Chromatin
Deoxyuracil Nucleotides - metabolism
DNA
DNA Replication
Eukaryota - cytology
Eukaryota - metabolism
Eukaryota - ultrastructure
Fluorescent Antibody Technique
Fluorescent antibody techniques
Fundamental and applied biological sciences. Psychology
Macronucleus
Microscopy, Electron
Molecular and cellular biology
Molecular genetics
Molecules
Monoclonal antibodies
Nucleotides
Replication
Shock heating
Cell culture
Protozoa
Replication
Ciliata
Macronucleus
Immunocytochemistry
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Summary:Isolated macronuclei from the hypotrichous ciliated protozoan Euplotes eurystomus incorporate biotinylated dUTP specifically into the replication band (RB) as detected with immunofluorescence, using rabbit anti-biotin antibodies followed by fluorescein-conjugated goat anti-rabbit IgG. When gold-conjugated goat anti-rabbit IgG was used in a preembedded reaction, subsequent immunoelectron microscopic analysis demonstrated that the biotinylated nucleotide appeared more concentrated in the rear zone of the RB, with almost no labeling in the forward zone. It was possible to use the immunofluorescent assay to establish that incorporation of biotinylated dUTP is inhibited by simultaneous addition of N-ethyl maleimide or aphidicolin, and by omission of any one of the other unlabeled dNTPs. In addition, prolonged heat shock of the intact cells, before lysis and in vitro assay, yielded markedly reduced incorporation. Comparison with published data on the in vivo incorporation of [3H]thymidine into Euplotes eurystomus RBs indicates the fidelity of the in vitro reaction.
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ISSN:0021-9525
1540-8140
DOI:10.1083/jcb.104.5.1125