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Updated: Aug 8, 2026
09:14
Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique
Published on: January 15, 2016
[Relation between DNA replication and neutral Mn 2+-dependent DNAse activity in chromatin]
Biokhimiia (Moscow, Russia)
|January 1, 1990
Insights
Serum stimulation in mouse fibroblasts promotes DNA synthesis, even when inhibited by aphidicolin and cytosine arabinoside. Antibodies targeting chromatin DNAase are internalized and accumulate in cell nuclei without affecting this DNA synthesis induction.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Context:
- Primary murine fibroblasts are utilized to investigate cellular responses to serum stimulation.
- DNA replication is studied under conditions of inhibition by specific agents like aphidicolin and cytosine arabinoside.
Purpose:
- To determine the effect of serum stimulation on DNA synthesis in the presence of inhibitors.
- To investigate the intracellular localization and persistence of antibodies within fibroblasts.
- To assess whether specific antibodies interfere with serum-induced DNA synthesis.
Summary:
- Serum stimulation was found to induce replicative DNA synthesis in primary murine fibroblasts, overcoming inhibition by aphidicolin and cytosine arabinoside.
- Direct immunofluorescence revealed that antibodies against chromatin DNAase penetrate cells, accumulating in the nucleus within 4 hours and persisting for at least 20 hours.
- Replacing these antibodies with bovine serum albumin or normal serum gamma-globulins did not impede the DNA synthesis induction by serum.
Impact:
- This study elucidates the mechanisms by which serum influences DNA replication in fibroblasts.
- It demonstrates the cellular uptake and nuclear localization of antibodies without functional interference.
- Provides insights into the regulation of DNA synthesis and the behavior of specific antibodies within the cellular environment.
Abstract:
In cultures of primary murine fibroblasts the 10% serum stimulates the replicative synthesis of DNA inhibited by aphidicolin and araC (cytosine arabinoside). Using direct immunofluorescence analysis, it was shown that antibodies penetrate inside the cells and after 4 hours are pooled in the nuclei, where they remain for another 20 hours. The substitution of antibodies against chromatin DNAase by bovine serum albumin of normal serum gamma-globulins does not interfere with the DNA synthesis induction.
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