Factors affecting the rearrangement efficiency of an Ig test gene.
A rearrangement test gene, pHRD, containing the mouse IgH enhancer and the metallothionein promoter, has previously been shown to rearrange efficiently after transfection into a pre-B cell line. Experiments are now reported that assess the requirements of the DNA substrate as well as of the transfected cells for efficient rearrangement. It was found that deletion of the metallothionein promoter or substitution of the IgH enhancer by the kappa enhancer did not affect rearrangement. However, deletion of the Ig enhancer reduced the efficiency. Transfection of pHRD into stable hybrids of pre-B cells and myeloma cells resulted in a high frequency of rearrangement only if certain myeloma chromosomes were lost. Furthermore, pHRD introduced into rearrangement incompetent myeloma cells upon subsequent cell fusion with pre-B cells was rearranged only very rarely and then apparently only immediately after cell fusion. Stable pre-B cell x myeloma hybrids that retained the critical myeloma chromosomes were found to have lost VDJ recombinase activity and transcripts of the RAG-1, RAG-2 and TdT genes. It is concluded that transcription, i.e., the copying of the DNA by polymerase, is probably not required for rearrangement, but that the rearrangement substrate must be in an "open" chromatin state, such as may be provided by transcriptional factors. Furthermore, the absence of rearrangement in myeloma cells is apparently due to the continued action of an inhibitor of rearrangement.
Engler
P
P
Department of Molecular Genetics and Cell Biology, University of Chicago, IL 60637.
Roth
P
P
Kim
J Y
JY
Storb
U
U
D000818:Animals; D001402:B-Lymphocytes; D015139:Blotting, Southern; D002874:Chromosome Mapping; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007136:Immunoglobulins / Q000235:genetics*; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D009101:Multiple Myeloma / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D007143:Immunoglobulin Heavy Chains; D007136:Immunoglobulins; D008668:Metallothionein