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 gamma-glutamylcysteine synthetase heavy subunit (gamma-GCSh) gene was investigated in COS7 cells transfected with hGH reporter constructs having successively deleted 5'-flanking sequence of the gamma-GCSh gene. Transcriptional activity was determined by the amounts of hGH secreted from the reporter constructs. Deletion of the sequence from -1,413 to -664 or -315 base pairs (bp) increased transcriptional activity from 100 to 138 or 136%. Further deletion from -315 to -241 bp, which contained an AP1 site, decreased transcriptional activity to 87%. Mutations introduced into the AP1 decreased transcriptional activity from 136 to 105%. These findings suggested that the AP1 increased transcriptional activity. When the sequence from -241 to -192 bp was deleted, transcriptional activity was restored from 87 to 128%. When this sequence was linked to the thymidine kinase promoter, it also decreased transcriptional activity by 38%. Deletion from -192 to -149, -116, or -108 bp did not significantly alter transcriptional activity. Further deletion of the GC-rich sequences from -108 to -70 and -28 bp dramatically decreased 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 transcriptional activity of the gamma-GCSh gene.
Tomonari
A
A
Pharmacology Division, National Cancer Center Research Institute, Tokyo, Japan.
Nishio
K
K
Kurokawa
H
H
Arioka
H
H
Ishida
T
T
Fukumoto
H
H
Fukuoka
K
K
Nomoto
T
T
Iwamoto
Y
Y
Heike
Y
Y
Itakura
M
M
Saijo
N
N
D001482:Base Composition; D001483:Base Sequence; D005796:Genes; D005721:Glutamate-Cysteine Ligase / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D012045:Regulatory Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D018808:Transcription Factor AP-1 / Q000235:genetics; D014158:Transcription, Genetic
D018808:Transcription Factor AP-1; D005721:Glutamate-Cysteine Ligase