Cloning and Functional Characterization of the Promoter Region of the Gene Encoding Human Adenylate Kinase Isozyme 3 Takafumi Noma, 1 Naoto Adachi, 2 and Atsushi Nakazawa Department of Biochemistry, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan Received September 30, 1999 The 5-flanking region of the gene encoding human adenylate kinase isozyme 3 was isolated and com- pared with that of the bovine AK3 gene previously characterized. Four conserved DNA sequences (ele- ments-a, -b, -c, and -d) were found in both the regions. The promoter activities were analyzed in HeLa cells using promoter-CAT reporter constructs. The proxi- mal promoter region (217 to 261), which contains three of four conserved elements, gave a maximum promoter activity. In a series of electrophoretic mobility-shift assays, DNA fragments and double- stranded oligodeoxyribonucleotides containing se- quences of the four conserved elements interacted with nuclear extracts of HeLa cells. The a-element con- tained the W-element, while the d-element, which had a high G  C content, was a novel regulatory cis- element distinct from the GC box. The b- and c-elements were homologous to each other and had a motif resembling downstream promoter element. Mu- tations of the c- and d-elements significantly reduced the promoter activity, indicating that the c- and d-elements in the AK3 promoter are crucial. These elements may also be involved in the transcriptional regulation of other TATA-less genes. © 1999 Academic Press Adenylate kinase (AK) is a ubiquitous enzyme that contributes to the homeostasis of the adenine nu- cleotide metabolism (1). Three isozymes (AK1, AK2, and AK3) have been characterized so far in verte- brates (2– 4). AK3, GTP:AMP phosphotransferase (EC 2.7.4.10), which catalyzes the reaction, Mg 2+ GTP + AMP ^ Mg 2+ GDP + ADP, is present in the mitochon- drial matrix of various tissues. The isozyme has been thought to play a role in the guanine nucleotide me- tabolism in mitochondria (5). The increase of AK3 mRNA level and AK3 enzyme activity were previously observed in rat skeletal muscle (4). In addition, AK3 protein was found to increase 10-fold during neural differentiation of P19 embryonal carcinoma cells (6). The induction of AK3 mRNA was also shown in re- sponse to hypoxia in HeLa cells depending on the pres- ence of hypoxia-inducible factor-1 (7). The 5'-flanking region of the bovine AK3 gene has a structural feature similar to those of housekeeping genes, in which the G + C content of the region is as high as 71%, and eight GC boxes were present (8). In this study, to further characterize the mechanism of transcriptional regulation of the AK3 gene, we iso- lated the 5'-flanking region of the human AK3 gene. Next, we analyzed the effect of deletion and mutation in the upstream regions on the promoter activity of the gene in HeLa cells. Thirdly, HeLa-cell nuclear factors were examined for binding to the AK3 promoter se- quences. The results suggest that nuclear proteins reg- ulate the transcription of the AK3 gene by binding to cis-regulatory elements, and that the elements may participate in the transcription initiation of TATA-less and Inr-less genes. MATERIALS AND METHODS Cloning of the human AK3 gene. A partial human genomic li- brary in EMBL3 containing DNA fragments digested with BamHI was screened using a 264-bp EcoRI-BstXI fragment that contains the 5' portion of a human AK3 cDNA (9) as a probe. A phage clone The nucleotide sequence of the human AK3 gene reported in this paper has been submitted to the DDBJ/EMBL/GenBank under Ac- cession No. D37992. Abbreviations used: AK, adenylate kinase; bp, base pair(s); CAT, chloramphenicol acetyltransferase; DPE, downstream promoter ele- ment; EMSA, electrophoretic mobility-shift assay; GCF, GC factor; HIP1, housekeeping initiator protein 1; Inr, initiator; kb, kilobase(s); nt, nucleotide(s); oligo, oligodeoxyribonucleotide(s); TAF, TBP- associated factor; TBP, TATA-binding protein. 1 To whom correspondence should be addressed. Fax: +81 836 22 2315. E-mail: tnoma@po.cc.yamaguchi-u.ac.jp. 2 Current address: Department of Neurosurgery, Yamaguchi Uni- versity School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan. Biochemical and Biophysical Research Communications 264, 990 –997 (1999) Article ID bbrc.1999.1616, available online at http://www.idealibrary.com on 990 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.