BIDIRECTIONAL TRANSCRIPTION FROM THE HUMAN-IMMUNOGLOBULIN V(H)6 GENE PROMOTER

The human immunoblobulin (Ig) heavy chain V(H)6 gene promoter contains an imperfect octamer (AgGCAAAT) and is not dependent on the Ig heavy chain enhancer for activity; reporter constructs containing this promo ter are very active in non-B cells. In experiments designed to charact erize regions upstream of the transcriptional start site that are impo rtant for promoter function, we produced a series of deletion construc ts, including one containing sequences between -74 and -146. Surprisin gly, this fragment had promoter activity in both orientations. Inspect ion of the V(H)6 promoter sequence indicated that there was a possible TATA box in the proper orientation upstream of the imperfect octamer. The -74 to -146 fragment functioned as a promoter in the reverse orie ntation in three B cell lines and in non-B (HeLa) cells, with a much h igher level of activity seen in the HeLa cells. To determine if the pr omoter could work in both directions simultaneously, reporter genes we re positioned up- and downstream of a V(H)6 promoter fragment. Reporte r gene activity was found for both genes in B cells and HeLa cells. Us ing a reverse transcriptase-polymerase chain reaction procedure (RT-PC R), we found a transcript corresponding to sequences upstream of the V (H)6 promoter in RNA from both the lymphoblastoid cell line ML-1, whic h actively transcribes the V(H)6 promoter, and the REH cell line, whic h does not. No transcripts were found in the KB epithelial cell line. Two or three mRNA 5' ends were found that mapped between -137 to -143 from the authentic V(H)6 transcription site, 31 -37 nucleotides upstea m of the putative TATA box. Inspection of the sequence upstream of the V(H)6 promoter demonstrated the presence of an open reading frame cap able of coding for 96 amino acids. The V(H)6 promoter represents the s econd Ig promoter with bidirectional activity.