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[mRNA of mouse plasmacytoma. Reverse transcription and translation in cell-free systems]
Abstract:
Total poly(A)-mRNA from polyribosomes of MOPC 21 mouse myeloma were investigated. Poly(A)-mRNA was released by two successive chromatography on oligo (dT)-cellulose. A 14S fraction of total poly(A)-mRNA was obtained and partially purified by sucrose gradient centrifigation followed by acrylamide gel electrophoresis. As estimated from the electrophoretic analysis, the 14S mRNA has three components, one of which appears to be 18S rRNA and two others--mRNAs with molecular weight of 5.2.10(5) and 3.8.10(5), respectively. Total poly(A)-mRNA and partially purified 14S mRNA were active when employed as a template in a reverse transcription and cell-free system from wheat germ. DNA complementary to the 14S mRNA was prepared with avian myeloblastosis virus RNA-dependent DNA polymerase. This cDNA was heterogeneous in size with the average size of about 800 nucleotides when analyzed by gel electrophoresis in 98% formamide. The maximal length was about 1100 nucleotides that consistent with full template length. About half of the translation product directed by the 14S mRNA migrated as mature L-chain Ig (upon polyacrylamide gel electrophoresis in sodium dodecylsulfate). The presented data suggested that 14S mRNA species contain mRNA L-chain Ig.
Insights
This study isolated 14S poly(A)-mRNA from mouse myeloma, finding it contains messenger RNA for L-chain immunoglobulin. This purified mRNA can be used to synthesize complementary DNA for further research.
Area of Science:
- Molecular Biology
- Immunology
Context:
- Investigated polyadenylated messenger RNA (poly(A)-mRNA) from MOPC 21 mouse myeloma polyribosomes.
- Poly(A)-mRNA was purified using oligo(dT)-cellulose chromatography.
Purpose:
- To isolate and characterize the 14S fraction of poly(A)-mRNA.
- To assess the translational activity and identify the coding potential of the 14S mRNA.
Summary:
- A 14S mRNA fraction was purified, containing components with molecular weights of 5.2x10^5 and 3.8x10^5, alongside 18S ribosomal RNA.
- This 14S mRNA was translated in a cell-free system, producing proteins, half of which corresponded to mature L-chain immunoglobulin.
- Complementary DNA (cDNA) synthesized from the 14S mRNA exhibited sizes consistent with full-length template, averaging 800 nucleotides.
Impact:
- The findings suggest that the 14S mRNA species isolated from mouse myeloma polyribosomes encodes L-chain immunoglobulin.
- This research provides a foundation for further studies on immunoglobulin gene expression and protein synthesis regulation.