Efficient expression of the yeast metallothionein gene in Escherichia coli.

The yeast metallothionein gene CUP1 was cloned into a bacterial expression system to achieve efficient, controlled expression of the stable, unprocessed protein product. The Escherichia coli-synthesized yeast metallothionein bound copper, cadmium, and zinc, indicating that the protein was functional. Furthermore, E. coli cells expressing CUP1 acquired a new, inducible ability to selectively sequester heavy metal ions from the growth medium.     

Regulation of the manganese-containing superoxide dismutase gene from fission yeast

The manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that dismutates a potentially toxic superoxide radical into hydrogen peroxide and dioxygen. To study the regulation of the Schizosaccharomyces pombe MnSOD gene, the 943 bp upstream region was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357, which resulted in the fusion plasmid pMS14. Restriction mapping and nucleotide sequencing confirmed its construction. The synthesis of beta-galactosidase from the fusion plasmid was induced by aluminum chloride, menadione, cadmium chloride, manganese chloride, and hydrogen peroxide. It was also induced by NO-generating S-nitroso-N-acetylpenicillamine (SNAP). However, cupric chloride and zinc chloride did not affect the synthesis of beta-galactosidase from the fusion plasmid. The beta-galactosidase synthesis appeared to be independent of the Pap1 protein. These results suggest that some metals, oxidative stress, and nitric oxide regulate the S. pombe MnSOD gene.     

The Neurospora crassa metallothionein gene. Regulation of expression and chromosomal location

The promoter region of the Neurospora crassa metallothionein gene contains no sequences which are similar to the mammalian or the yeast metal responsive elements (Münger, K., Germann, U. A., and Lerch, K. (1985) EMBO J. 4, 2665-2668). We therefore studied the regulation of expression of the N. crassa metallothionein gene in response to different metal ions (Cu2+, Cd2+, Zn2+, Co2+, and Ni2+) by Northern analysis. Only copper led to the induction of metallothionein mRNA. In N. crassa cultures inoculated and grown in copper-supplemented media, metallothionein mRNA appeared during the late logarithmic growth period (about 30 h after inoculation) and was detectable for a time period of more than 30 h. In response to copper shock, however, rapidly increasing amounts of metallothionein mRNA were detected within minutes after copper administration at any time in vegetatively growing mycelia of N. crassa. Maximum levels were detected about 1 h after addition of copper to the medium. The half-life time of the mRNA was estimated as 2.5 h. The amounts of copper metallothionein reach a maximum level at 3 h after induction and thereafter remain constant. The rapid induction by copper ions of metallothionein mRNA and metallothionein together with the remarkable stability of the native protein intracellularly suggest that this protein serves an important homeostatic role in the copper metabolism in this fungus. The structural gene of N. crassa metallothionein has been located on chromosome VI using restriction fragment-length polymorphisms as genetic markers.     

The CUP2 gene product regulates the expression of the CUP1 gene, coding for yeast metallothionein.

The yeast CUP1 gene codes for a copper-binding protein similar to metallothionein. Copper sensitive cup1s strains contain a single copy of the CUP1 locus. Resistant strains (CUP1r) carry 12 or more multiple tandem copies. We isolated 12 ethyl methane sulfonate-induced copper sensitive mutants in a wild-type CUP1r parental strain, X2180-1A. Most mutants reduce the copper resistance phenotype only slightly. However, the mutant cup2 lowers resistance by nearly two orders of magnitude. We cloned CUP2 by molecular complementation. The smallest subcloned fragment conferring function was approximately 2.1 kb. We show that CUP2, which is on chromosome VII, codes for or controls the synthesis or activity of a protein which binds the upstream control region of the CUP1 gene on chromosome VIII. Mutant cup2 cells produced extremely low levels of CUP1-specific mRNA, with or without added copper ions and lacked a factor which binds to the CUP1 promoter. Integrated at the cup2 site, the CUP2 plasmid restored the basal level and inducibility of CUP1 expression and led to reappearance of the CUP1-promoter binding factor. Taken collectively, our data establish CUP2 as a regulatory gene for expression of the CUP1 metallothionein gene product.     

Regulation of the gene encoding glutathione synthetase from the fission yeast

The fission yeast cells that contained the cloned glutathione synthetase (GS) gene showed 1.4-fold higher glutathione (GSH) content and 1.9-fold higher GS activity than the cells without the cloned GS gene. Interestingly, gamma-glutamylcysteine synthetase activity increased 2.1-fold in the S. pombe cells that contained the cloned GS gene. The S. pombe cells that harbored the multicopy-number plasmid pRGS49 (containing the cloned GS gene) showed a higher level of survival on solid media with cadmium chloride (1 mM) or mercuric chloride (10 microM) than the cells that harbored the YEp357R vector. The 506 bp upstream sequence from the translational initiation point and N-terminal 8 amino acid-coding region were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pUGS39. Synthesis of beta-galactosidase from the fusion plasmid pUGS39 was significantly enhanced by cadmium chloride and NO-generating S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SN). It was also induced by L-buthionine-(S,R)-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase (GCS). We also found that the expression of the S. pombe GS gene is regulated by the Atf1-Spc1-Wis1 signal pathway.     

Expression of the Drosophila melanogaster metallothionein genes in yeast

The metallothionein system in Drosophila melanogaster is composed of two genes, Mto and Mtn, that code for distinctly different proteins. In order to compare the properties of Mto and Mtn, we transformed yeast with several fusion plasmids. The Mto and Mtn cDNAs, when placed under the control of CUP1 or PGK promoters, can confer a copper-resistance phenotype to copper-hypersensitive cells. Both Mto and Mtn proteins can be characterized in extracts from transformed yeast cells.     

X-ray absorption studies of yeast copper metallothionein

The local structures of the metal sites in copper metallothionein from Saccharomyces cerevisiae have been investigated by x-ray absorption spectroscopy at the copper and sulfur K edges. Analysis of the EXAFS (extended x-ray absorption fine structure) data indicates that each copper is trigonally coordinated to sulfur at a distance of 2.23 A. Cu-Cu interactions at 2.7 and 3.9 A have also been tentatively identified. Sulfur K edge data are compatible with cysteinyl thiolates bridging each of the eight Cu(I) ions. The data support a model for the copper cluster in yeast metallothionein consisting of a Cu8S12 core. EXAFS data on two specifically engineered carboxyl-terminal truncated mutants reveal that the copper coordination in the mutants is similar to that observed in the wild-type protein.     

Amplification of a gene for metallothionein by tandem repeat in a strain of cadmium-resistant yeast cells

Abstract In a cadmium-resistant strain of Saccharomyces cerevisiae , cells are protected against cadmium toxicity by the production of large amounts of cadmium-binding metallothionein, as occurs similarly in a copper-resistant strain. The apoprotein of the metallothionein is encoded by the CUP1 gene on chromosome VIII. The CUP1 gene is present as 8–10 copies in the cadmium-resistant strain as a result of tandem repeat of a 2.0-kb fragment of DNA that includes CUP1 , while the wild-type strain contains only a single copy of CUP1 . In the cadmium-resistant strain, some evidence for elongation of chromosome VIII with variations in length (maximum to 200 kb) was obtained. However, the elongation was not due to the tandem repeats of the CUP1 -containing region.