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Purification and characterization of DNA polymerases from the plasmacytoma MOPC 104E and Abelson murine leukemia
Abstract:
RNA-dependent DNA polymerases of intracisternal A particles from the mouse plasma cell tumor MOPC 104E and of Abelson murine leukemia virus (A-MuLV) were isolated from particle preparations by Nonidet P40 and ultrasonic treatment and purified by column chromatography on DEAE-cellulose and phosphocellulose, followed by centrifugation in linear sucrose gradients. Both DNA polymerases were very similar in their elution patterns from phospho and DEAE-cellulose, template specificities, requirements for optimum activity and inactivation by anti-(reverse transcriptase) antiserum. They are associated with ribonuclease H activity. For molecular weight determinations, antibody-precipitated enzymes were bound to staphylococcal-protein-A-Sepharose, solubilized and run on dodecylsulfate/polyacrylamide gels. Their apparent molecular weight was estimated to be 80000.
Insights
Researchers purified and characterized RNA-dependent DNA polymerases from mouse plasma cell tumors and Abelson murine leukemia virus. Both enzymes showed similar properties, including association with ribonuclease H activity and an estimated molecular weight of 80,000.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Intracisternal A particles and Abelson murine leukemia virus (A-MuLV) are retroviral elements implicated in cellular processes.
- RNA-dependent DNA polymerases (reverse transcriptases) are key enzymes in retroviral replication and are also found in cellular systems.
Purpose of the Study:
- To isolate and purify RNA-dependent DNA polymerases from mouse plasma cell tumor MOPC 104E and A-MuLV.
- To compare the biochemical and immunological properties of these two DNA polymerases.
Main Methods:
- Enzyme isolation using Nonidet P40 and ultrasonic treatment.
- Purification via DEAE-cellulose and phosphocellulose chromatography, followed by sucrose gradient centrifugation.
- Molecular weight determination using dodecylsulfate/polyacrylamide gel electrophoresis after antibody precipitation.
Main Results:
- Both DNA polymerases exhibited similar elution profiles on ion-exchange columns.
- Template specificities and optimal activity requirements were comparable between the two enzymes.
- Both enzymes were inactivated by anti-(reverse transcriptase) antiserum and associated with ribonuclease H activity.
- The apparent molecular weight for both enzymes was estimated to be 80,000.
Conclusions:
- The RNA-dependent DNA polymerases from MOPC 104E intracisternal A particles and A-MuLV share significant biochemical and immunological similarities.
- These findings suggest a commonality in the structure or function of these retroviral enzymes.