Molecular analysis of membrane immunoglobulin-negative variants

Area of Science:

• Immunology
• Molecular Biology
• Cell Biology

Background:

• WEHI 279.1 is a mouse B-cell lymphoma producing both membrane and secreted immunoglobulin M (IgM).
• Immunoselection yielded variants lacking membrane IgM (mIgM-), often due to loss of mu heavy chain synthesis.
• Four mIgM- mutants were selected for molecular analysis to understand IgM regulation.

Purpose of the Study:

• To investigate the molecular basis for the loss of membrane immunoglobulin M (mIgM) expression in WEHI 279.1 B-cell lymphoma variants.
• To determine if defects in mu heavy chain gene arrangement, RNA synthesis, or processing underlie the mIgM- phenotype.
• To explore potential complementation of mIgM defects using somatic cell hybridization.

Main Methods:

• Immunoselection to isolate mIgM- variants.
• DNA analysis to assess mu gene arrangement.
• RNA analysis to quantify and characterize mu heavy chain transcripts.
• Somatic cell hybridization to test for complementation of defects.

Main Results:

• One mutant had a large deletion of the expressed mu gene; three had intact mu gene arrangements.
• All mutants produced low levels (10-30%) of cytoplasmic mu RNA, but not of appropriate size for membrane (mu m) or secreted (mu s) forms.
• Non-deletion mutants transcribed mu RNA at wild-type levels, suggesting defects in RNA stability, processing, or nuclear transport.
• Somatic cell hybrids showed partial complementation of the mIgM- defect by the P3X fusion partner.

Conclusions:

• The loss of mIgM in WEHI 279.1 variants is primarily due to post-transcriptional defects in mu heavy chain RNA processing or stability, rather than gene rearrangement.
• Elevated levels of non-functional 'sterile' mu RNA transcripts may be present in some mutants.
• Complementation of mIgM defects is possible, indicating potential for restoring normal B-cell receptor expression.