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Isolation of Trypanosoma brucei variant specific antigen mRNA by immunoprecipitation of polysomes
Abstract:
The mRNA encoding the variant specific antigen of Trypanosoma brucei has been prepared by immunoprecipitation of polysomes. Polysomes carrying the variant specific antigen account for approx. 3% of the total polysomes. The mRNA thus produced is active in the mRNA dependent rabbit reticulocyte lysate in vitro protein synthesis system and directs the synthesis of a polypeptide of 60 000 daltons which co-migrates both with 125I-labelled purified variant specific antigen and with antigen immunoprecipitated from reticulocyte lysate charged with total polyadenylated mRNA from the same clone. The mRNA is being used both to prepare cDNA clones and to prepare high specific radioactivity cDNA to be used to screen a gene bank for clones containing variant specific antigen coding sequences.
Insights
Researchers isolated messenger RNA (mRNA) for a Trypanosoma brucei variant specific antigen using polysome immunoprecipitation. This mRNA successfully directed protein synthesis in vitro, producing the correct antigen, aiding gene cloning efforts.
Area of Science:
- Molecular Biology
- Parasitology
- Immunology
Background:
- Trypanosoma brucei evades the host immune system through antigenic variation.
- Understanding the molecular mechanisms of variant specific antigen expression is crucial for developing control strategies.
Purpose of the Study:
- To isolate and characterize the messenger RNA (mRNA) encoding the Trypanosoma brucei variant specific antigen.
- To utilize the isolated mRNA for generating complementary DNA (cDNA) for gene cloning and screening.
Main Methods:
- Polysome immunoprecipitation was employed to isolate mRNA associated with the variant specific antigen.
- In vitro protein synthesis using a rabbit reticulocyte lysate system was performed to assess mRNA activity.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation were used to analyze synthesized proteins.
Main Results:
- Approximately 3% of total polysomes were found to carry the variant specific antigen mRNA.
- The isolated mRNA directed the synthesis of a 60,000-dalton polypeptide in vitro.
- The synthesized polypeptide co-migrated with purified variant specific antigen, confirming its identity.
Conclusions:
- The study successfully isolated functional mRNA for the Trypanosoma brucei variant specific antigen.
- This mRNA is a valuable tool for generating cDNA clones and screening gene banks to identify antigen-coding sequences.
- The findings facilitate further research into antigenic variation and potential therapeutic targets.
