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Purification of 14S messenger RNA of immunoglobulin light chain that codes for a possible light-chain precursor
Abstract:
Polysomes released from microsomes of MOPC 41 mouse myeloma were used to prepare a poly(A)-containing fraction of RNA by chromatography on poly-(dT)-cellulose. From that fraction, a 14S RNA species was purified to a single peak by successive sucrose gradient centrifugations, followed by acrylamide gel electrophoresis. The RNA has an apparent molecular weight of 380,000 (1100 nucleotides), as estimated from the electrophoretic analyses. In a reticulocyte lysate this RNA directs the synthesis of a protein that migrates more slowly in sodium dodecylsulfate-acrylamide gels than does the light chain secreted by the same tumor. This difference in migration corresponds to a size difference appropriate for polypeptide chain about 20 amino acids longer than the light chain. The tryptic peptides of this protein correspond to those of the secreted light chain, except for the presence of two additional peptides from the product synthesized in vitro and for the absence of one light-chain peptide. The purified RNA is, therefore, the mRNA of the light chain, and it seems to code for a precursor protein slightly larger than the light chain. From the estimated size of the 14S mRNA, it appears that only 65% of the RNA is translated.
Insights
Researchers isolated messenger RNA (mRNA) coding for mouse myeloma light chains. This mRNA directs the synthesis of a precursor protein, slightly larger than the final light chain, indicating a post-translational modification.
Area of Science:
- Molecular Biology
- Protein Synthesis
- RNA Analysis
Background:
- Mouse myeloma cells (MOPC 41) are a model for studying immunoglobulin synthesis.
- Polysomes are complexes of mRNA and ribosomes involved in protein translation.
- Polyadenylated RNA (poly(A)-containing RNA) is often associated with mature mRNA.
Purpose of the Study:
- To isolate and characterize the messenger RNA (mRNA) responsible for synthesizing the light chain protein in MOPC 41 mouse myeloma.
- To investigate the nature of the protein product directed by the purified mRNA in a cell-free system.
Main Methods:
- Isolation of polysomes from mouse myeloma microsomes.
- Chromatography on poly(dT)-cellulose to obtain poly(A)-containing RNA.
- Purification of a 14S RNA species using sucrose gradient centrifugation and acrylamide gel electrophoresis.
- In vitro protein synthesis using a reticulocyte lysate system.
- Analysis of synthesized protein by SDS-acrylamide gel electrophoresis and tryptic peptide mapping.
Main Results:
- A 14S RNA species was purified, estimated to have a molecular weight of 380,000 (1100 nucleotides).
- This RNA directed the synthesis of a protein larger than the secreted myeloma light chain.
- Tryptic peptide analysis revealed the synthesized protein contained unique peptides not found in the light chain, and lacked one light chain peptide.
- The data suggests the mRNA codes for a precursor protein to the light chain.
Conclusions:
- The purified 14S RNA is the messenger RNA (mRNA) for the myeloma light chain.
- The mRNA appears to code for a precursor polypeptide that is slightly larger than the secreted light chain.
- Approximately 65% of the 14S mRNA sequence is translated into protein.