Abstract
We have established a cell-free system, derived from a human B-cell lymphoma, in which immunoglobulin kappa light chain gene promoters are both accurately transcribed and regulated in a cell-type-specific manner. Thus, accurate transcription from the T1 kappa light chain gene promoter was much more efficient in B-cell extracts than in HeLa cell extracts, whereas control promoters (adenovirus major late and histone H2B) were transcribed equally well in either extract. More important, the increased kappa light chain gene transcription in B-cell extracts was dependent upon upstream sequences (containing the conserved decanucleotide element) previously shown to be necessary for B-cell-specific transcription in vivo; in contrast, removal of these sequences had no effect on the low level of kappa transcription in HeLa extracts. The maximal level of upstream sequence-mediated transcription was dependent upon template topology. These studies show that there is at least one B-cell-specific factor that stimulates transcription from purified DNA templates, and they further suggest that the in vivo action of the factor(s) on other components of the transcription machinery is direct rather than indirect (e.g., via the maintenance of an open chromatin structure). The cell-free system described here should facilitate both purification and functional studies of the B-cell-specific factor(s).