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Updated: Aug 17, 2026
Isolation of Precursor B-cell Subsets from Umbilical Cord Blood
Published on: April 16, 2013
Identification of an intracellular precursor to DNA excreted by human lymphocytes
Abstract:
Phytohemagglutinin-stimulated human peripheral blood lymphocytes in vitro synthesize DNA that is excreted into the culture medium. When such cells are pulse-labeled with [3H]thymidine during the peak of DNA synthesis on day 3 of culture, then cultured for 3 more days in the absence of isotope, labeled DNA moves slowly into the Hirt supernatant cell fraction from the pellet fraction containing chromosomal DNA,and then into the culture medium. The number of copies of excreted DNA sequences in the Hirt pellet fraction was determined for lymphocytes harvested on days 3,4, and 6 after stimulation and compared to the number found in resting lymphocyte DNA and in placenta DNA. While resting lymphocyte and placenta DNAs contain one to two copies of sequences similar to excreted DNA per haploid genome, stimulated lymphocytes on days 3 and 4 of culture contain 3- to 4-fold more copies; by day 6 of culture, stimulated lymphocytes contain only 1- to 2-fold more copies than resting lymphocytes. Thus, phytohemagglutinin induces lymphocytes to selectively replicate several copies of a limited portion of their genome, copies which are then excreted into the culture medium. As determined by reassociation kinetics analysis, a high-molecular-weight DNA fraction from the Hirt supernatant contains sequences found in excreted DNA. This DNA may represent an intermediate formed prior to release of excreted sequences from the cells.
Insights
Phytohemagglutinin (PHA) stimulation causes human lymphocytes to replicate specific DNA sequences. These replicated DNA copies are then excreted into the culture medium, suggesting a novel cellular process.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Human peripheral blood lymphocytes, when stimulated by phytohemagglutinin (PHA) in vitro, exhibit DNA synthesis.
- This synthesized DNA is subsequently released into the surrounding culture medium.
- The fate and origin of this excreted DNA are not fully understood.
Purpose of the Study:
- To investigate the replication and excretion dynamics of DNA synthesized by PHA-stimulated human lymphocytes.
- To determine the copy number of excreted DNA sequences in stimulated versus resting lymphocytes and placenta.
- To identify potential intermediates in the excretion process.
Main Methods:
- Pulse-labeling of lymphocytes with [3H]thymidine during peak DNA synthesis.
- Fractionation of cellular DNA using Hirt lysis (pellet and supernatant).
- Quantification of DNA sequence copy numbers via reassociation kinetics analysis.
- Analysis of high-molecular-weight DNA fractions from the Hirt supernatant.
Main Results:
- Stimulated lymphocytes selectively replicate several copies of a limited portion of their genome.
- The copy number of excreted DNA sequences increases 3- to 4-fold in lymphocytes on days 3 and 4 post-stimulation compared to resting cells.
- By day 6, the copy number decreases but remains elevated compared to resting lymphocytes.
- A high-molecular-weight DNA fraction in the Hirt supernatant contains sequences found in excreted DNA, potentially representing an excretion intermediate.
Conclusions:
- Phytohemagglutinin induces selective DNA replication and subsequent excretion in human lymphocytes.
- This process involves an increase in specific DNA sequence copy numbers within stimulated cells.
- The excreted DNA may play a role in intercellular communication or immune response modulation.
