IDENTIFICATION OF CIS-ACTING DNA ELEMENTS OF THE HUMAN GAMMA-GLUTAMYLCYSTEINE SYNTHETASE HEAVY SUBUNIT GENE

Transcriptional activity of the 5'-flanking sequence of the human gamm a-glutamylcysteine synthetase heavy subunit (gamma-GCS(h)) gene was in vestigated in COS7 cells transfected with hGH reporter constructs havi ng successively deleted 5'-flanking sequence of the gamma-GCS(h) gene. Transcriptional activity was determined by the amounts of hGH secrete d from She reporter constructs, Deletion of the sequence from -1,413 t o -664 or -315 base pairs (bp) increased transcriptional activity from 100 to 138 or 136%. Further deletion from -315 to -241 bp, which cont ained all AP1 site, decreased transcriptional activity to 87%. Mutatio ns introduced into the AP1 decreased transcriptional activity from 136 to 105%. These findings suggested that the AP1 increased transcriptio nal activity. When the sequence from -241 to -192 bp was deleted, tran scriptional activity was restored from 87 to 128%. When this sequence was linked to the thymidine kinase promoter, it also decreased transcr iptional activity by 38%. Deletion from -192 to -149, -116, or -108 bp did not significantly alter transcriptional activity. Further deletio n of the GC-rich sequences from -108 to -70 and -28 bp dramatically de creased transcriptional activity from 135 to 87 and 34%, respectively. These findings indicate that multiple DNA elements, especially those in the proximal GC-rich sequences, are involved in the regulation of t ranscriptional activity of the gamma-GCS(h) gene. (C) 1997 Academic Pr ess.