We present evidence that two distinct regions of the DNA upstream from the mouse metallothionein-I gene contain metal-responsive regulatory sites. This result was obtained by analyzing a systematic series of deletion, insertion, duplication, and clustered point mutations introduced into cultured cells on a simian virus 40 plasmid vector. The two upstream regions contain a duplicated evolutionarily conserved DNA sequence. While either upstream region is sufficient to confer heavy metal responsiveness, both are required to give maximal levels of induced transcription.The metallothioneins (MTs) are small cysteine-rich proteins that tightly bind heavy metals. They are present in a broad range of eukaryotes, from yeast to man, and are expressed in many different tissues and cell types. Exposure to heavy metals results in a rapid increase in MT mRNA levels and protein synthesis in both cultured cells and whole animals. This homeostatic regulatory mechanism plays a critical role in protecting cells against toxic ions, such as cadmium and mercury, and may also be involved in the metabolism of essential elements such as zinc and copper (reviewed in ref. 1). The metal response, which occurs in the presence of protein synthesis inhibitors, can be attributed largely if not exclusively to increased transcription rates (2-5).What MT gene DNA sequences are involved in the rapid transcriptional response to heavy metals? We have shown previously that a cloned mouse MT-I gene, containing 2000 base pairs (bp) of 5' flanking DNA, retains its ability to be induced by cadmium when introduced into cultured monkey kidney cells on simian virus 40 (SV40) viral and plasmid vectors (4). Appropriate regulation has also been observed for mouse and human MT and MT fusion genes carried on episomal bovine papilloma virus vectors (6, 7), introduced into cultured mouse or rat cells by transformation (5, 8), microinjected into mouse eggs (9), or integrated into the genome in transgenic mice (10). To more precisely localize and characterize the MT regulatory sequences, we have constructed a systematic series of 3' deletions, 5' deletions, internal deletions, duplications, insertions, and clustered point mutations in the mouse MT-I gene. These were introduced into cultured cells on an SV40 plasmid vector and analyzed both for their efficiency of transcription and for their ability to be induced by cadmium. We have also compared the 5' flanking DNA sequences of the mouse MT-I gene with three functional human MT genes. Our results show that the metalresponsive elements lie in at least two distinct regions of the 5' flanking DNA. These regions share a conserved nucleotide sequence that may serve as a recognition signal for cellular regulatory factors. MATERIALS AND METHODSRecombinant plasmids were constructed and characterized by standard methods (11). The starting pML-SV40-MT construct, JYMMT(E), was described previously (4). The Escherichia coli galactokinase-SV40 transcription unit was obtained from pSVK105A, a derivative of pSVK carrying a ...

Metallothioneins (MTs), small cysteine-rich proteins, bind to and are inducible by heavy metals such as zinc, cadmium and copper. Recent gene-transfer and mutagenesis experiments have elucidated cis-acting DNA sequences involved in this form of regulation, but nothing is known about the trans-acting factors that interact with the control sequences or how such interactions influence the rate of transcription. We report here the detection of cellular factors involved in the cadmium induction of the mouse MT-1 gene by an in vivo competition assay. We show that at least one-class of these cellular factors acts by a positive regulatory mechanism depending on the same region of the 5' flanking DNA required for maximal transcription.

During bacteriophage T7 infection a phage-specified RNA polymerase transcribes the late phage genes in two temporal classes (class II and class III). In this report, we show that the purified phage polymerase discriminates between the class II and class III promoters in vitro as a function of variables that alter the stability of the DNA helix. These variables include ionic strength, temperature, and the presence of denaturing agents such as dimethyl sulfoxide. In general, initiation at the class II promoters is preferentially inhibited as helix stability is increased. Conditions required for the establishment of salt-resistant transcription complexes by the T7 RNA polymerase have been determined; the establishment of stable complexes at the class II promoters requires the synthesis of a longer nascent RNA transcript than does formation of such complexes at the class III promoters. A comparison of the nucleotide sequences of several class II and class III promoters suggests certain features that may be responsible for the different responses of these promoters to helix destabilization. The conservation of structural features that are peculiar to the class II or class III promoters indicates that these features are important in regulation of T7 transcription in vivo. Experiments which bear on the physiological significance of these features are discussed.

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