Cloning and functional characterization of the promoter region of the gene encoding human adenylate kinase isozyme 3.
The 5'-flanking region of the gene encoding human adenylate kinase isozyme 3 was isolated and compared with that of the bovine AK3 gene previously characterized. Four conserved DNA sequences (elements-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 proximal 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 sequences of the four conserved elements interacted with nuclear extracts of HeLa cells. The a-element contained 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. Mutations 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.
Noma
T
T
Department of Biochemistry, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi, 755-8505, Japan. tnoma@po.cc.yamaguchi-u.ac.jp
Adachi
N
N
Nakazawa
A
A
D000263:Adenylate Kinase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*
D007527:Isoenzymes; D000263:Adenylate Kinase