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[Insertion of mouse kappa light chain immunoglobulin gene sequences into a bacterial plasmid]
Abstract:
The 14S mRNA from MOPC 173 tumour has been transcribed into cDNA by AMV DNA polymerase and converted into a double stranded form by Escherichia coli DNA polymerase I. After addition of oligo-dG tracts at the 3'-OH ends by calf thymus terminal transferase this DNA was hybridized to an E. coli plasmid (pCR1) to which oligo-dG tracts had been similarly added. Circular molecules resulting from GC base pairing have been used to transform C 600 E. coli cells and to confer kanamycine resistance. Several recombinants have been obtained containing the V+C regions and the C or V region alone. These recombinant molecules are being used to analyse the translocation of V and C genes and to purify V and C genes from DNA of germ line cells and of differentiated tumour.
Insights
Researchers created recombinant DNA from MOPC 173 tumor mRNA to study gene translocation. This method allows for the purification of immunoglobulin V and C genes from various cell types.
Area of Science:
- Molecular Biology
- Immunogenetics
- Biotechnology
Background:
- The MOPC 173 tumor produces 14S mRNA, a key component in immunoglobulin production.
- Understanding gene structure and expression in tumors is crucial for cancer research.
Purpose of the Study:
- To develop a method for creating recombinant DNA from tumor-derived mRNA.
- To analyze the translocation of immunoglobulin variable (V) and constant (C) genes.
- To enable the purification of V and C genes from germ line and tumor DNA.
Main Methods:
- Transcription of 14S mRNA into cDNA using AMV DNA polymerase.
- Conversion to double-stranded DNA using Escherichia coli DNA polymerase I.
- Addition of oligo-dG tracts and hybridization to an E. coli plasmid (pCR1).
- Transformation of E. coli cells and selection for kanamycin resistance.
Main Results:
- Successful generation of recombinant DNA molecules containing V+C regions, or V or C regions alone.
- Demonstration of GC base pairing for circular molecule formation.
- Obtained several recombinant clones with specific immunoglobulin gene segments.
Conclusions:
- The developed method is effective for creating recombinant DNA from tumor mRNA.
- These recombinant molecules are valuable tools for studying immunoglobulin gene translocation.
- The technique facilitates the purification of specific immunoglobulin genes from different cellular sources.
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