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Characterization of an RNA-directed DNA polymerase found in association with murine intracytoplasmic A-particles
Abstract:
An RNA-directed DNA polymerase was found to be associated with intracytoplasmic A-particles from DBA/2 mouse leukemia cells. The enzyme activity was detected after disrupting the purified particles with 2 M NaCl-20 mM dithiothreitol. The presence of a divalent cation and all four deoxyribonucleoside triphosphates was essential for this enzyme activity. The enzyme had a clear preference for Mg2+ over Mn2+. Cesium sulfate isopycnic gradient centrifugation of the DNA product synthesized in the actinomycin D-containing reaction revealed the presence of DNA-RNA hybrid. Furthermore, the purified DNA product was found to hybridize with RNA isolated from A-particles. These observations strongly indicate that the endogenous A-particle RNA serves as the template for the DNA polymerase.
Insights
Researchers discovered an RNA-directed DNA polymerase in mouse leukemia A-particles. This enzyme uses the A-particle RNA as a template to synthesize DNA, a key finding in retrovirology research.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Intracytoplasmic A-particles are found in DBA/2 mouse leukemia cells.
- The presence and function of associated enzymes within these particles were previously unclear.
Purpose of the Study:
- To identify and characterize enzymes associated with intracytoplasmic A-particles.
- To determine the template used by any identified DNA polymerase.
Main Methods:
- Purification of intracytoplasmic A-particles from mouse leukemia cells.
- Disruption of particles and assay for enzyme activity in the presence of deoxyribonucleoside triphosphates and divalent cations.
- Cesium sulfate isopycnic gradient centrifugation to analyze synthesized DNA.
- Hybridization studies between synthesized DNA and A-particle RNA.
Main Results:
- An RNA-directed DNA polymerase activity was detected in disrupted A-particles.
- Enzyme activity required divalent cations (Mg2+ preferred over Mn2+) and all four deoxyribonucleoside triphosphates.
- Synthesized DNA formed a DNA-RNA hybrid and hybridized with A-particle RNA.
Conclusions:
- The identified enzyme is an RNA-directed DNA polymerase.
- Endogenous A-particle RNA serves as the template for this DNA polymerase.
- This suggests a potential role for reverse transcription in the replication or function of these particles.