PubMed1987
During the maturation of an immune response, some IgM bearing B lymphocytes differentiate into IgG secreting cells. Similarly, a subset of normal B cells cultured in the presence of a mitogen will develop into IgG synthesizing cells, many of which continue to express IgM. The experiments described in this paper utilize such IgG3 expressing B cells present in mitogen-stimulated cultures to address the continuing controversy over the molecular mechanism(s) allowing simultaneous expression of IgM and isotypes other than IgD. Nascent transcripts were labelled in vitro in order to determine whether the C mu gene was still transcribed in these cells. If no mu transcription was detected, then this would suggest that the mu chain protein, in cells expressing both IgM and IgG3, must be derived from translation of long-lived mu mRNA. Alternatively, detection of continued mu gene transcription would provide direct evidence for alternate processing of pre-mRNA transcripts encoding both mu and gamma gene sequences. The membrane IgG3+ (mIgG3+) cells were isolated from mitogen-stimulated cultures by staining and then sorting on a Fluorescence Activated Cell Sorter (FACS). Prior to examining mu gene transcription in these cells, it was necessary to ascertain the purity of the sorted population. Accordingly, restaining of the sorted IgG3+ cells showed only a minor contamination by mIgM+IgG3- cells. Additionally, the majority of the mIgG3+ cells were also mIgM+. Most importantly, it was then demonstrated that the IgG3 detected on the cells was intrinsic to them rather than cytophilically adsorbed. By biosynthetic labelling, the sorted IgG3+ population was found to also actively secrete both IgM and IgG3. Examination of transcription showed that the IgG3+ cells were indeed transcribing the mu exons and were doing so at about 50% the level of an equal number of unseparated cells. It was determined that this level of C mu transcription is significantly higher than that which could be derived from the contaminating cells. Thus, it is clear that at least some IgG3 expressing cells have not deleted the C mu gene and therefore the gamma 3 mRNA in a portion of the cells expressing both IgM and IgG3 must be derived from pre-mRNA transcripts which contain both C mu and C gamma 3 sequences.(ABSTRACT TRUNCATED AT 400 WORDS)