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Transcription of the Saccharomyces cerevisiae metallothionein gene CUP1 is induced in response to high environmental levels of copper. Induction requires the ACE1 gene product, which binds to specific sites in the promoter region of the CUP1 gene. In this study, we found that deleting the entire coding sequence of the ACE1 gene resulted in a decrease in basal-level transcription of CUP1 to low but detectable levels and conferred a copper-sensitive phenotype to the cells. We have isolated a gene, designated ACE2, which when present on a high-copy-number plasmid suppresses the copper-sensitive phenotype of an ace1-deletion strain. The presence of multiple copies of the ACE2 gene enhanced expression of an unlinked CUP1-lacZ fusion integrated in the yeast genome and resulted in an increase in the steady-state levels of CUP1 mRNA in an ace1-deletion background. A large deletion of the coding region of the genomic copy of ACE2 resulted in a decrease in steady-state levels of CUP1 mRNA, indicating that ACE2 plays a role in regulating basal-level expression of CUP1. The ACE2 open reading frame encodes a polypeptide of 770 amino acids, with putative zinc finger structures near the carboxyl terminus. This protein is 37% identical to the SWI5 gene product, an activator of HO gene transcription in S. cerevisiae, suggesting that ACE2 and SWI5 may have functional similarities.
Mol Cell Biol 1991 Jan
PMID:ACE2, an activator of yeast metallothionein expression which is homologous to SWI5. 198 41

In the yeast Saccharomyces cerevisiae, transcription of the metallothionein gene CUP1 is induced by copper and silver. Strains with a complete deletion of the ACE1 gene, the copper-dependent activator of CUP1 transcription, are hypersensitive to copper. These strains have a low but significant basal level of CUP1 transcription. To identify genes which mediate basal transcription of CUP1 or which activate CUP1 in response to other stimuli, we isolated an extragenic suppressor of an ace1 deletion. We demonstrate that a single amino acid substitution in the heat shock transcription factor (HSF) DNA-binding domain dramatically enhances CUP1 transcription while reducing transcription of the SSA3 gene, a member of the yeast hsp70 gene family. These results indicate that yeast metallothionein transcription is under HSF control and that metallothionein biosynthesis is important in response to heat shock stress. Furthermore, our results suggest that HSF may modulate the magnitude of individual heat shock gene transcription by subtle differences in its interaction with heat shock elements and that a single-amino-acid change can dramatically alter the activity of the factor for different target genes.
Mol Cell Biol 1991 Mar
PMID:Heat shock transcription factor activates transcription of the yeast metallothionein gene. 199 89

Single crystals of pseudoazurin, one of the blue copper proteins produced by methylotrophic bacterium Methylobacterium extorquens AM1, have been obtained by the method of vapor diffusion with ammonium sulfate as a precipitant at pH 8.0. Crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions of a = 52.619(4) A, b = 63.280(6) A, c = 35.133(4) A. The asymmetric unit includes one molecule of pseudoazurin (Vm = 2.18 A3/dalton). The crystals are so stable against X-ray irradiation that diffraction intensities of the native crystal up to 1.68 A resolution could be collected from only one crystal. Among the many heavy-metal reagents examined, uranyl acetate gave an effective isomorphous derivative.
J Mol Biol 1991 Mar 05
PMID:Preliminary crystallographic study of a pseudoazurin from methylotrophic bacterium, Methylobacterium extorquens AM1. 200 2

The structural gene, nirS, for the respiratory nitrite reductase (cytochrome cd1) from Pseudomonas stutzeri was identified by (i) sequencing of the N-terminus of the purified protein and partial sequencing of the cloned gene, (ii) immunoscreening of clones from a lambda gt11 expression library, (iii) mapping of the transposon Tn5 insertion site in the nirS mutant strain MK202, and (iv) complementation of strain MK202 with a plasmid carrying the insert from an immunopositive lambda clone. A mutation causing overproduction of cytochrome c552 mapped on the same 8.6 kb EcoRI fragment within 1.7 kb of the mutation affecting nirS. Two mutations affecting nirD, which cause the synthesis of an inactive cytochrome cd1 lacking heme d1, mapped 1.1 kb apart within a 10.5 kb EcoRI fragment contiguous with the fragment carrying nirS. Nir- mutants of another type that had low level synthesis of cytochrome cd1, had Tn5 insertions within an 11 kb EcoRI fragment unlinked to the nirS+ and nirD+ fragments. Cosmid mapping provided evidence that nirS and nirD, and the previously identified gene cluster for nitrous oxide respiration are closely linked. The nirS gene and the structural gene for nitrous oxide reductase, nosZ, are transcribed in the same direction and are separated by approximately 14 kb. Several genes for copper processing are located within the intervening region.
Mol Gen Genet 1991 Feb
PMID:Close linkage in Pseudomonas stutzeri of the structural genes for respiratory nitrite reductase and nitrous oxide reductase, and other essential genes for denitrification. 200 66

A 3.3 kb fragment of yeast genomic DNA was isolated by screening a genomic library constructed in the high copy number 2 micron plasmid YEp351 vector for clones capable of enhancing the degree of resistance of Saccharomyces cerevisiae strain MW3070-8B to cadmium. The insert contained two complete copies of the CUP1 gene open reading frame (183 bp), including the upstream promoter sequences (450 bp) with two conserved metal responsive cis-acting elements. Northern analysis showed that addition of cadmium (0.02 microM) or copper (50 microM) to overnight liquid cultures of yeast induced expression of CUP1 transcripts from both chromosomal and plasmid-borne gene copies. The cloned 3.3 kb DNA in a high copy number plasmid restored copper resistance to the sensitive strain LS70-3B delta, deleted for the CUP1 gene (cup 1 delta), but failed to restore cadmium resistance. Thus, CUP1 gene expression in yeast appears to be influenced differently by cadmium and copper ions. Resistance to heavy metal poisoning resulted from enhanced gene product levels attributable to amplification of the CUP1 gene as well as to increased transcriptions. Two distinct gene product levels mediate cadmium and copper resistance; a higher gene product level was required to confer cadmium resistance.
Mol Gen Genet 1991 Mar
PMID:Multicopy CUP1 plasmids enhance cadmium and copper resistance levels in yeast. 201 34

As part of a comparative study on the binding of different metals and anions by human lactoferrin, we have prepared and crystallized: (1) dicupric lactoferrin with Cu2+ and carbonate in each site (Cu2Lf); and (2) a lactoferrin complex with Cu2+ and carbonate in one site, and Cu2+ and oxalate in the other (Cu2oxLf). Crystals of Cu2Lf are orthorhombic: a = 155.9, b = 97.0, c = 56.0 A, space-group P2(1)2(1)2(1); those of Cu2oxLf are also orthorhombici a = 155.9, b = 97.1, c = 56.2 A, space-group P2(1)2(1)2(1). Both are isomorphous with diferric human lactoferrin, Fe2Lf. Diffractometer data to 2.6 A and 2.5 A have been collected for Cu2Lf and Cu2oxLf, respectively. Difference maps show that the main effect of substitution of Cu2+ for Fe3+ is a small shift (0.5 to 1.0 A) in the metal position in each site. For Cu2oxLf the oxalate ion is found to be accommodated in the C-lobe, bound to copper in a bidentate mode, causing only small local changes, in the positions of adjacent Arg and Tyr side-chains.
J Mol Biol 1991 May 20
PMID:Preliminary crystallographic studies of copper(II)- and oxalate-substituted human lactoferrin. 203 52

Plastocyanin can be detected in Synechocystis sp. PCC 6803 when 3 microM copper is added to the growth medium, BG-11. The plastocyanin gene (petE) was cloned from a genomic lambda EMBL 3 library by screening with the petE gene from Anabaena sp. PCC 7937. The Synechocystis 6803 petE gene is present as a single copy and, as deduced from the DNA sequence, encodes a precursor protein of 126 amino acids. The predicted 29 amino acid transit peptide shows substantial homology to the Anabaena 7937 transit peptide, thought to direct the plastocyanin precursor to the thylakoid lumen. Putative promoter sites -16 and -38 base pairs from the start of the petE gene have been identified. The deduced amino acid sequence has the greatest homology (61%) to the green alga Scenedemus obliquus plastocyanin. Despite the lower homology, the copper binding residues and certain aromatic residues remain highly conserved. Northern hybridization analysis indicates that the Synechocystis sp. PCC 6803 petE gene is not transcriptionally regulated since the accumulation of petE mRNA appears to be independent of the copper concentration in the growth media. The possibility of an additional polypeptide needed to facilitate the electron transfer from plastocyanin to P700+ is also discussed.
Plant Mol Biol 1990 Oct
PMID:Copper-induced expression, cloning, and regulatory studies of the plastocyanin gene from the cyanobacterium Synechocystis sp. PCC 6803. 212 38

CUP2 is a copper-dependent transcriptional activator of the yeast CUP1 metallothionein gene. In the presence of Cu+ and Ag+) ions its DNA-binding domain is thought to fold as a cysteine-coordinated Cu cluster which recognizes the palindromic CUP1 upstream activation sequence (UASc). Using mobility shift, methylation interference, and DNase I and hydroxyl radical footprinting assays, we examined the interaction of wild-type and variant CUP2 proteins produced in Escherichia coli with the UASc. Our results suggest that CUP2 has a complex Cu-coordinated DNA-binding domain containing different parts that function as DNA-binding elements recognizing distinct sequence motifs embedded within the UASc. A single-amino-acid substitution of cysteine 11 with a tyrosine results in decreased Cu binding, apparent inactivation of one of the DNA-binding elements and a dramatic change in the recognition properties of CUP2. This variant protein interacts with only one part of the wild-type site and prefers to bind to a different half-site from the wild-type protein. Although the variant has about 10% of wild-type DNA-binding activity, it appears to be completely incapable of activating transcription.
Mol Cell Biol 1990 Sep
PMID:A single amino acid change in CUP2 alters its mode of DNA binding. 216 39

To define the import pathway for apoiso-1-cytochrome c in vivo, the coding region for bacterial chloramphenicol acetyltransferase (CAT) or yeast copper metallothionein (CuMT) was fused to the carboxy terminus of the apoiso-1-cytochrome c (iso-1) coding region. When the resulting iso-1/CAT and iso-1/CuMT fusion proteins were individually expressed in Saccharomyces cerevisiae, they were specifically targeted to the mitochondria and protected from trypsin digestion. Although iso-1/CAT was accessible to heme modification, it remained membrane associated because of the folded conformation of the CAT domain. A small deletion disrupting CAT structure resulted in the translocation of the resulting fusion protein, iso-1/CAT delta, to the intermembrane space, where it functioned efficiently in respiratory electron transfer. Similarly, iso-1/CuMT was heme modified and nearly identical to iso-1 in its ability to support respiratory growth, indicating that the CuMT domain was compatible with translocation to the IMS. Inclusion of copper in the growth medium, which converts the loosely structured apo-CuMT to a tightly folded holo-CuMT, inhibited both heme attachment and respiratory growth without affecting mitochondrial targeting. Thus, by altering the folded conformation of the reporter moiety of these fusion proteins, it was possible to differentiate between those molecules arrested at the mitochondrial targeting step of the cytochrome c import pathway and those translocated to the intermembrane space. By replacing the heme-binding cysteine residues with serines, this system was used to demonstrate that the import requirement for heme attachment operated at the level of membrane translocation and not on mitochondrial targeting in vivo.
Mol Cell Biol 1990 Nov
PMID:In vivo expression and mitochondrial targeting of yeast apoiso-1-cytochrome c fusion proteins. 217 84

Three nuclear factors, the Ah receptor, XF1, and XF2, bind sequence specifically to the Ah response elements or xenobiotic response elements (XREs) of the cytochrome P450IA1 (P450c) gene. The interactions of these factors with the Ah response element XRE1 were compared by three independent methods, methylation interference footprinting, orthophenanthroline-Cu+ footprinting, and mobility shift competition experiments, using a series of synthetic oligonucleotides with systematic alterations in the XRE core sequence. These studies established the following (i) all three factors interact sequence specifically with the core sequence of XRE1; (ii) the pattern of contacts made with this sequence by the Ah receptor are different from those made by XF1 and XF2; and (iii) although XF1 and XF2 can be distinguished by the mobility shift assay, the sequence specificities of their interactions with XRE1 are indistinguishable. Further characterization revealed the following additional differences among these three factors: (i) XF1 and XF2 could be extracted from nuclei under conditions quite different from those required for extraction of the Ah receptor; (ii) XF1 and XF2 were present in the nuclei of untreated cells and did not respond to polycyclic compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-napthoflavone, while nuclear Ah receptor was undetectable in untreated cells and rapidly increased in response to TCDD; (iii) inhibition of protein synthesis did not affect the TCDD-induced appearance of the Ah receptor but substantially decreased the constitutive activities of XF1 and XF2, suggesting that the Ah receptor must be present in untreated cells in an inactive form that can be rapidly activated by polycyclic compounds, while the constitutive expression of XF1 and XF2 depends on the continued synthesis of a relatively unstable protein; (iv) the receptor-deficient and nuclear translocation-defective mutants of the hepatoma cell line Hepa1, which are known to lack nuclear Ah receptor, expressed normal levels of XF1 and XF2, suggesting that the former factor is genetically distinct from the latter two; and (v) a divalent metal ion, probably Zn2+, is known to be an essential cofactor for the Ah receptor but was not required for the DNA-binding activities of XF1 and XF2. Together, these findings indicate that the Ah receptor is distinct from XF1 and XF2, while the latter two activities may be related. Because the DNA-binding domains of these three factors overlap substantially, their binding to XREs is probably mutually exclusive, which suggests that the interplay of these factors at Ah response elements may be important to the regulation of CYP1A1 gene transcription. The results of preliminary transfection experiments with constructs harboring XREs upstream of the chloramphenicol acetyltransferase gene driven by a minimal simian virus 40 promoter are presented that are consistent with this hypothesis.
Mol Cell Biol 1990 Dec
PMID:Multiple DNA-binding factors interact with overlapping specificities at the aryl hydrocarbon response element of the cytochrome P450IA1 gene. 217 7


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