Expression and regulation of a human metallothionein gene carried on an autonomously replicating shuttle vector.

Karin, Michael; Cathala, Guy; Nguyen-Huu, M C

doi:10.1073/pnas.80.13.4040

Cited by 84 publications

(53 citation statements)

References 32 publications

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“…We cannot rule out the possibility that the 800 bp exogenous MT-IIA promoter used in this study does not contain all relevant regulatory elements; however this promoter has previously been shown to be regulated in a manner similar to the endogenous gene (Karin et al, 1983(Karin et al, , 1984. MT genes, including MT-IIA, have been shown to be regulated by methylation (Jahroudi et al, 1990;Stennard et al, 1994).…”

Section: Discussionmentioning

confidence: 91%

“…By supplying or removing zinc MTs can activate or inactivate various zinc-binding proteins (Udom and Brady, 1980;Zeng et al, 1991), and thus regulate a variety of cellular processes. MT proteins also confer protection against non-essential heavy metals (Karin et al, 1983) and may act directly as antioxidants (Thornalley and VasaÂ k, 1985). MT-IIA expression is induced by various hormones and cytokines, as well as by DNA damaging agents (reviewed in KaÈ gi, 1991).…”

Section: Introductionmentioning

confidence: 99%

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Downregulation of metallothionein-IIA expression occurs at immortalization

Duncan

Reddel

1999

Oncogene

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Section: Discussionmentioning

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Downregulation of metallothionein-IIA expression occurs at immortalization

Duncan

Reddel

1999

Oncogene

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“…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)(10)(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)(13)(14)(15)(16)(17)(18).…”

mentioning

confidence: 99%

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

Stuart¹,

Searle²,

Chen³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

368

216

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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...

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“…In mammals, induction of MT by metals is due to transcriptional activation of MT genes by heavy metal ions (Beach and Palmiter, 1981;Durnam and Palmiter, 1981;Karin et al, 1983;Karin et al, 1985). In this study, CAT activity in Drosophila S2 cells transfected with the plasmid MT-CAT was induced by 10-100 nM cadmium.…”

Section: Discussionmentioning

confidence: 90%

“…MT genes encode low molecular weight, cystein-rich metal binding proteins found in a wide variety of organisms including bacteria, fungi, insects, plants, fish, rodents and humans (Kagi and Kojima, 1979), and are thought to play several important roles in metal homeostasis (Kagi and Schaffer, 1988) and in protecting against metal toxicities (Beach and Palmiter, 1981;Karin et al, 1983;Karin et al, 1985). Using transgenic flies bearing the MJ-lacZ fusion gene, the regulation of the MT promoter activity during Drosophila development was examined.…”

Section: Discussionmentioning

confidence: 99%