Bidirectional transcription from the human immunoglobulin VH6 gene promoter

• Published in: Nucleic acids research 1994-03, Vol.22 (5), p.861-868
• Main Authors: Sun, Zijie, Kitchingman, Geoffrey R.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; B-Lymphocytes - metabolism; Base Sequence; Biological and medical sciences; Cell Line, Transformed; DNA; Fundamental and applied biological sciences. Psychology; HeLa Cells; Humans; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Variable Region - genetics; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Polymerase Chain Reaction; Promoter Regions, Genetic; Transcription, Genetic; Transcription. Transcription factor. Splicing. Rna processing; Tumor Cells, Cultured
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/22.5.861

The human immunoblobulln (lg) heavy chain VH6 gene promoter contains an imperfect octamer (AgGCAAAT) and is not dependent on the Ig heavy chain enhancer for activity; reporter constructs containing this promoter are very active in non-B cells. In experiments designed to characterize regions upstream of the transcrlptional start site that are important for promoter function, we produced a series of deletion constructs, including one containing sequences between − 74 and −146. Surprisingly, this fragment had promoter activity in both orientations. Inspection of the VH6 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 orientation in three B cell lines and in non-B (HeLa) cells, with a much higher level of activity seen in the HeLa cells. To determine if the promoter could work in both directions simultaneously, reporter genes were positioned up- and downstream of a VH6 promoter fragment. Reporter gene activity was found for both genes in B cells and HeLa cells. Using a reverse transcrlptase-polymerase chain reaction procedure (RT-PCR), we found a transcript corresponding to sequences upstream of the VH6 promoter in RNA from both the lymphoblastold cell line ML-1, which actively transcribes the VH6 promoter, and the REH cell line, which 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 VH6 transcription site, 31 – 37 nucleotides upsteam of the putative TATA box. Inspection of the sequence upstream of the VH6 promoter demonstrated the presence of an open reading frame capable of coding for 96 amlno acids. The VH6 promoter represents the second Ig promoter with bidirectional activity.