Vol. 159, No. 1, 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS February 28, 1989 Pages 343-348 ANALYSIS OF THE HUMAN ADRENODOXIN PROMOTER: EVIDENCE FOR ITS ACTIVITY Bon-chu Chung *, Char-Chang Lai, Cheng-Hui Lin, and Chi-Yao Chang Institute of Molecular Biology Academia Sinica Nankang, Taipei, Taiwan, Republic of China, 11529 Received January 23, 1989 Adrenodoxin is an iron-sulfur protein serving as an electron transfer intermediate in the mitochondrial cytochrome P450 system. To study its transcriptional regulation we construct a human adrenodoxin genomic clone which includes 333 bp of DNA upstream of the mRNA start site. This DNA contains a TATA box and two GC boxes. When we place it in front of the CAT reporter gene and transfect it into the recipient cell lines, it directs transcription of the CAT gene. This DNA in reverse orientation does not show any transcriptional activity. This promoter element functions in three mammalian cell lines: JEG-3, COS-1, and Y-l. 0 1989 Academic Press, Inc. The steroid hydroxylase systems in the mitochondria contain the terminal cytochrome P450, adrenodoxin, and adrenodoxin reductase. Adrenodoxin is an iron-sulfur protein that transfers electrons in the hydroxylase system. It is expressed abundantly in steroidogenic tissues and in lower amount in liver and kidney (1). The expression of adrenodoxin is stimulated by CAMP (1). This regulated expression is controlled at the transcriptional level (2). The human adrenodoxin gene is located on chromosome 11 and its pseudogenes on chromosome 20 (3). In order to understand the mechanism of transcriptional regulation, we cloned the human adrenodoxin gene and sequenced all its four exons and the 5’-flanking region (4). We now report the promoter activity of this gene. MATERIALS AND METHODS Cell Lines and Transfection Assays Y-l cells and human choriocarcinoma cell line JEG-3 were obtained from B. P. Schimmer and J. F. Strauss III. Cells were plated onto 6 cm *To whom correspondence should be addressed. 0006-291x/89 $1.50 343 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.