Newborn mouse DNA was digested with a restriction endonuclease EcoRI and concentrated with respect to ribosomal RNA sequences by an RPC-5 column.DNA fragments of 14-17 kilobases in length, most probably containing promoter region of the ribosomal RNA gene, were used for cloning with λgt WES•λB as a vector using an in vitro packaging technique. Several clones containing 18S rRNA sequences were obtained. One of the clones which was transferred to a plasmid pBR322 (designated as pMrEL-1) was 14 kilobases in length, having only a part of 18S rRNA sequence. These results strongly suggest that this fragment carries a promoter region of the ribosomal RNA gene.
The molecular mechanism of any tumor marker expression may shed a light on the mechanism of the particular tumorigenesis. This idea in mind, we have been pursuing the mechanism of specific induction of the placental type glutathione transferase (GST-P) gene during hepatocarcinogenesis of the rat. Making use of advanced technologies of molecular biology including proteomic analysis, gene cloning and production of specific transgenic rats etc., we were able to identify the enhancer and the activator proteins responsible for this tumor marker expression. Negative regulatory regions and modulatory proteins were also found. The overview of this long range study and the future outlook of the problem will be discussed. (Communicated by Kumao TOYOSHIMA, M.J.A.)
The Pi-class glutathione S-transferases (GSTs) play pivotal roles in the detoxification of xenobiotics, carcinogenesis and drug resistance. The mechanisms of regulation of these genes during drug induction and carcinogenesis are yet to be elucidated. Recently, Nrf2 (NF-E2-related factor 2; a bZip-type transcription factor) knockout mice were shown to display impaired induction of Pi-class GST genes by drugs. It is known that the mouse Pi-class GST gene GST-P1 is expressed predominantly in the male liver, and is regulated by androgen. To determine whether Nrf2 and the androgen receptor regulate GST-P1 directly, we analysed the molecular mechanism of activation of this gene by these factors. The promoter of the GST-P1 gene was activated markedly by Nrf2 in transient transfection analyses. Gel mobility shift assay and footprinting analyses revealed three Nrf2 binding sites: one at the proximal and two at distal elements, located at positions -59, -915 and -937 from the cap site. The fifth intron of the GST-P1 gene contains the androgen-responsive region. Multiple androgen receptor binding sites are clustered within a 500 bp region of this intron. The whole fragment contains a minimum of seven androgen receptor binding sites, which collectively display strong androgen-dependent enhancer activity. However, on division into small fragments containing two or three elements each, individual enhancer activities were dramatically decreased. This suggests that multiple elements work synergistically as a strong androgen-responsive enhancer. Our findings indicate that Nrf2 and the androgen receptor directly bind to and activate the mouse GST-P1 gene.
