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Gamma heavy chain disease in man: translation and partial purification of mRNA coding for the deleted protein
Abstract:
Lymphoid cells obtained from the peripheral blood of a patient with heavy chain disease have been established in long-term culture. They continue to produce a protein antigenically identical to the deleted gamma3 heavy chain disease protein found in the patient's serum. The availability of the cell line has made it possible to analyze the mRNA coding for this protein. The primary in vitro translation product is 1500-2000 daltons larger than the polypeptide portion of the cytoplasmic or secreted protein and has methionine at the amino terminus. The mRNA sediments at 15.5 S on sucrose gradients and therefore appears to be smaller than the 17S message coding for normal-sized mouse gamma chains. It contains a base sequence that codes for a hydrophobic amino-terminal peptide not found in the cytoplasmic protein. There was no evidence for the synthesis of translatable light chain message by these cells. The present data suggest that this protein results from a primary somatic genetic event that gave rise to a cell product bearing a normal aminoterminus sensitive to limited proteolytic digestion. The serum protein thus appears to begin in the hinge region but, in fact, contains a normal heavy chain initiation site.
Insights
Researchers established a cell line from a patient with heavy chain disease, which produces the disease-specific protein. Analysis revealed unique mRNA characteristics, suggesting a specific genetic event in B cell development.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Established a long-term culture of lymphoid cells from a patient diagnosed with heavy chain disease.
- Confirmed the continuous production of a protein antigenically identical to the patient's serum gamma3 heavy chain disease protein.
Purpose of the Study:
- To analyze the messenger RNA (mRNA) encoding the disease-specific protein using the established cell line.
- To investigate the molecular basis of the aberrant protein production in heavy chain disease.
Main Methods:
- Cell culture and protein production analysis.
- In vitro translation assays.
- Sucrose gradient sedimentation of mRNA.
- Amino-terminal sequencing of translation products.
Main Results:
- The primary in vitro translation product was larger (1500-2000 daltons) than the mature protein, with methionine at the amino terminus.
- The mRNA sedimented at 15.5S, smaller than the typical 17S mRNA for normal gamma chains.
- The mRNA contained a sequence for a hydrophobic amino-terminal peptide absent in the cytoplasmic protein.
- No translatable light chain mRNA was detected in these cells.
Conclusions:
- The findings suggest the protein arises from a primary somatic genetic event.
- The aberrant protein likely results from a normal heavy chain initiation site followed by limited proteolytic digestion.
- The serum protein, despite appearing to start in the hinge region, originates from a normal heavy chain initiation site.