High levels of transcription driven by a 400 bp segment of the human G6PD promoter.
A 2850-base-pair-long DNA segment containing the transcriptional start site of the human X-linked gene coding for the housekeeping enzyme glucose-6-phosphate dehydrogenase has been fused to the reporter chloramphenicol acetyl transferase gene. This fusion was introduced into three different mammalian cell lines (HepG2, CVI and HeLa). Strong expression was detected and transcription initiated at the natural start site. By deletion analysis, we determined that a 436 base pair region of this promoter is sufficient for full expression of the chimaeric construct and therefore the remainder of the CpC island surrounding the transcriptional start site and differentially methylated in the active and inactive X chromosome is not necessary for transcriptional activity in this assay. Further sequence deletions from -436 to -116 base pairs decrease the promoter-directed chloramphenicol acetyl transferase activity. Site-directed mutagenesis of a TATA-like sequence present in the G6PD promoter shows that this site is required for a correct start of transcription but not for determining the level of gene expression.
Ursini
M V
MV
International Institute of Genetics and Biophysics, CNR, Naples, Italy.
Scalera
L
L
Martini
G
G
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009710:Nucleotide Mapping; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D014162:Transfection; D014982:Xenopus laevis
D012333:RNA, Messenger; D004247:DNA; D005954:Glucosephosphate Dehydrogenase; D015500:Chloramphenicol O-Acetyltransferase