A 12-base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene.

Abstract

To define DNA sequences involved in mouse metallothionein-I (MT-I) gene promoter function and metal regulation, we fused the 5' flanking sequences of the MT-I gene to the coding sequences of a viral thymidine kinase (TK) gene. A series of 5' deletion, 3' deletipn, linker-scanning, and internal deletion mutants of the MT-I promoter was constructed and assayed by microinjection into mouse eggs. The results indicate that at least two related promoter elements can confer some metal regulation independently. Those mutations that had the most severe effect on regulation impinge on a 12-basepair conserved sequence that is repeated several times within the mouse MT-I and other MT promoters. To test the regulatory function of this sequence, it was synthesized as a pair of complementary oligonucleotides and inserted into the promoter of the TK gene. A single insertion of this sequence conferred limited metal regulation onto the TK promoter, whereas a construct with two separate inserts was regulated as efficiently as the MT-I promoter.Metallothionein (MT) genes are expressed in most animal tissues and cell lines (1-3). They are transcriptionally regulated by a variety of heavy metals (3, 4), by glucocorticoids (5-7), and by a hormone liberated in response to inflammation (8, 9). Several MT genes have now been cloned and shown to retain expression and regulation after transfer into heterologous cells (6, 9-11). The MT promoter and 5' flanking sequences have been used to confer metal, steroid, and/or inflammatory response to a variety of structural genes that were transferred into cells or animals (12-18). The location of elements involved in these regulatory functions is being actively pursued by the reverse genetic approach of in vitro mutagenesis and gene transfer. Short DNA sequences thought to be involved in both metal (13,16,18) and glucocorticoid (16) regulation have been proposed based on deletion mutagenesis; however, precise sequence requirements for efficient interaction with regulatory proteins have not been defined. In this study, we show that at least two independent promoter elements mediate the metal inducibility of the mouse MT-I gene and that a synthetic copy of one of these elements confers metal regulation to a heterologous gene. MATERIALS AND METHODS -C-C-C-T-T-T-G-C-G-C-C-C-G-A 3' and 5' G-A-T-C-T-C-G-G-G-C-G-C-A-A-A-G 3' (provided by J. Habener). Linear molecules were purified by electrophoresis on agarose gels, eluted, and heated to 68°C in 50 mM NaCl. The 13 nucleotide "sticky ends" were annealed at 20°C overnight before transformation and cloning. The oligonucleotide insertion regenerated the BamHI site at only one end, which allows the orientation of the insert to be determined.As copies of the reference plasmid, pMT-,Bgal, were injected (in -2 pl) into the male pronuclei of -20 fertilized mouse eggs. The eggs were then divided into two groups and incubated for 22 hr in the presence or absence of 50 ,M CdSO4. Aphidicolin (2 ,uM), an inhibitor of DNA synthesis, was included in the medium t...

The acute promyelocytic leukemia PML-RARα protein induces hepatic preneoplastic and neoplastic lesions in transgenic mice

“…We constructed a recombinant plasmid, MT-PMLRARa, that places the PML-RARa type L cDNA (De The , et al, 1991) under the control of the mouse metallothionein promoter (Stuart, et al, 1984) (Figure 1a). This strategy was based on previous ®ndings that the same promoter can drive the expression of the BCR-ABL protein in the myeloid cell compartment of transgenic mice leading to the development of leukemia (Heisterkamp et al, 1990).…”

“…To generate the MT-PML-RARa construct, a 250 bp fragment of the mouse metallothionein promoter, nucleotides 7185 to +65, (Stuart et al, 1984) was joined to the 4.6 kb PML-RARa type L cDNA (De The et al, 1991a) including the RARa 3' untranslated region and the two presumed polyadenylation signals (GigueÁ re et al, 1987). The transgene was microinjected into pronuclei of fertilized (C57BL/66SJL/J) F2 hybrid eggs.…”

Activation and repression of transcription by homoeodomain-containing proteins that bind a common site

“…CAT reaction time courses and extract mixing experiments indicated that little or no inhibitory activity was present in low activity extracts (data not shown). β-galactosidase (β-gal) activity (from responders 3 and 4 and hsp82LacZ reference gene used with responders 1, 2 and 5) was determined by fluorimetric assay 58 . Drosophila ADH activity was determined as described 59 .…”

Metallothionein RNA levels in HL‐60 cells Effect of cadmium, differentiation, and protein kinase C activation

“…MT gene expression is predominantly regulated at the level of transcription [5][6][7][8][9]. Though the mechanism of regulation is poorly understood, rapid induction occurs upon exposure to metals [5] and, in mammalian cells, to various circulating factors such as glucocorticoid hormones [6], ainterferon [7], and interleukin-1 and other mediators of the inflammatory response [8,9].…”

“…Though the mechanism of regulation is poorly understood, rapid induction occurs upon exposure to metals [5] and, in mammalian cells, to various circulating factors such as glucocorticoid hormones [6], ainterferon [7], and interleukin-1 and other mediators of the inflammatory response [8,9]. It has been suggested that a common pathway for MT induction involves protein kinase C (PKC) activation since MT levels increase in serum-starved cells after treatment with growth factors and TPA, agents which function in part through PKC activation [10,11].…”