Addition of copper to yeast cells leads to the induction of a low molecular weight, cysteine-rich protein that binds copper. This protein, termed copper chelatin or thionein, is related to the metallothionein family of proteins that are induced in response to cadmium and zinc in vertebrate cells. We have determined the structure of the yeast copperbinding protein by DNA sequence analysis of the gene. Although the 6573-dalton yeast protein is substantially divergent from vertebrate metallothioneins, the arrangement of 12 cysteine residues, which is a hallmark of metal-binding proteins, is partially conserved. We analyzed the regulatory DNA sequence of the gene by fusing it with the Escherichia coli galactokinase gene and assaying the levels of enzyme activity in yeast in response to copper. The transcriptional activation has a specific requirement for copper. Zinc, cadmium, and gold were unable to regulate the galactokinase activity. The yeast copper metallothionein regulatory sequences represent a previously unreported class of yeast promoter that is regulated by copper.Methallothioneins (MTs) are thought to play a central role in the protection against heavy metal toxicity and zinc homeostasis (see ref. 1 for a review). Recent interest in the molecular biology of MT stems from the observation that the genes for mammalian MT are regulated by heavy metals as well as by glucocorticoids (2-3). MT genes from several different mammalian systems such as mouse, Chinese hamster, monkey, and human have been cloned and sequenced (4-7).Although the precise mechanism of MT gene activation by heavy metals has not been elucidated, a DNA sequence upstream of the 5' end of the mouse MT I gene, which is responsible for cadmium inducibility, has been identified (8, 9 Recently we cloned a copper-inducible gene from a CUpir strain and showed that it protects a cupP strain from copper toxicity (14). The restriction map of this clone indicated that it is identical to the CUP] gene previously cloned and characterized by Fogel and Welch (12). To provide a basis for understanding the function and regulation of this gene, we determined the primary structure of the coding region and flanking sequences. We show that it encodes a protein with both similarities to and differences from the MTs of higher eukaryotes. We refer to this protein as copper metallothionein (Cu-MT) because it belongs to the metallothionein family of proteins as defined in ref. Fig. 4). Growth 3332The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The infectious yeast Candida albicans progresses through two developmental programs which involve differential gene expression, the bud-hypha transition and high-frequency phenotypic switching. To understand how differentially expressed genes are regulated in this organism, the promoters of phase-specific genes must be functionally characterized, and a bioluminescent reporter system would facilitate such characterization. However, C. albicans has adopted a nontraditional codon strategy that involves a tRNA with a CAG anticodon to decode the codon CUG as serine rather than leucine. Since the luciferase gene of the sea pansy Renilla reniformis contains no CUGs, we have used it to develop a highly sensitive bioluminescent reporter system for C. albicans. When fused to the galactose-inducible promoter of GAL1, luciferase activity is inducible; when fused to the constitutive EF1␣2 promoter, luciferase activity is constitutive; and when fused to the promoter of the white-phase-specific gene WH11 or the opaque-phase-specific gene OP4, luciferase activity is phase specific. The Renilla luciferase system can, therefore, be used as a bioluminescent reporter to analyze the strength and developmental regulation of C. albicans promoters.Reporter genes which code for bioluminescent gene products, like the luciferases, have provided a very rapid method for analyzing the regulation of gene expression (4) and a highly sensitive method for single-cell analysis (38). Recently, we used the firefly luciferase gene (FLUC) fused in frame with the phase-regulated WH11 gene of Candida albicans as a reporter to functionally characterize the 5Ј upstream regulatory region of WH11 (29), but the analyses were restricted to Northern (RNA) blots because we were unable to identify a translation product of the firefly luciferase, either through enzyme activity or as a FLUC-related peptide in Western blots (immunoblots; unpublished observations). The lack of a detectable translation product was most likely due to a nontraditional codon strategy adopted by C. albicans and related species (19,22,23). These organisms use a tRNA with a CAG anticodon to decode the codon CUG as serine, while most organisms use CAG to decode the codon CUG as leucine. Recently, it was demonstrated that the traditional leucine isoacceptor tRNA for CUG from Saccharomyces cerevisiae is toxic to C. albicans (13). Furthermore, direct determination of the amino acid sequences of peptides derived from three aspartyl proteinases of C. albicans confirmed the presence of serine instead of leucine at nucleotide positions containing the CUG codon (38). FLUC contains nine in-frame CUG codons, making it highly unlikely that a functional luciferase could be expressed in C. albicans. In order to circumvent this codon problem, we have developed a reporter system for C. albicans using the luciferase gene RLUC of the sea pansy Renilla reniformis, which contains no CUG codons in its open reading frame (ORF). We have fused the Renilla luciferase gene to a number of promoters of C. albicans a...

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) activity in Saccharomyces cerevisiae is allosterically regulated by CTP product inhibition. Amino acid residue Glu 161 in the URA7-encoded and URA8-encoded CTP synthetases was identified as being involved in the regulation of these enzymes by CTP product inhibition. The specific activities of the URA7-encoded and URA8-encoded enzymes with a Glu 161 3 Lys (E161K) mutation were 2-fold greater when compared with the wild-type enzymes. The E161K mutant URA7-encoded and URA8-encoded CTP synthetases were less sensitive to CTP product inhibition with inhibitor constants for CTP of 8.4-and 5-fold greater, respectively, than those of their wild-type counterparts. Cells expressing the E161K mutant enzymes on a multicopy plasmid exhibited an increase in resistance to the pyrimidine poison and cancer therapeutic drug cyclopentenylcytosine and accumulated elevated (6 -15-fold) levels of CTP when compared with cells expressing the wild-type enzymes. Cells expressing the E161K mutation in the URA7-encoded CTP synthetase exhibited an increase (1.5-fold) in the utilization of the Kennedy pathway for phosphatidylcholine synthesis when compared with control cells. Cells bearing the mutation also exhibited an increase in the synthesis of phosphatidylcholine (1.5-fold), phosphatidylethanolamine (1.3-fold), and phosphatidate (2-fold) and a decrease in the synthesis of phosphatidylserine (1.7-fold). These alterations were accompanied by an inositol excretion phenotype due to the misregulation of the INO1 gene. Moreover, cells bearing the E161K mutation exhibited an increase (1.6-fold) in the ratio of total neutral lipids to phospholipids, an increase in triacylglycerol (1.4-fold), free fatty acids (1.7-fold), and ergosterol ester (1.8-fold), and a decrease in diacylglycerol (1.3-fold) when compared with control cells. These data indicated that the regulation of CTP synthetase activity by CTP plays an important role in the regulation of phospholipid synthesis.CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADPforming)) is a cytosolic-associated glutamine amidotransferase that catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP (1, 2). This enzyme plays an essential role in the synthesis of all membrane phospholipids in eukaryotic cells (3, 4). Its reaction product CTP is the direct precursor of the activated, energyrich phospholipid pathway intermediates CDP-DG 1 (5), CDPcholine (6), and CDP-ethanolamine (6) (Fig. 1). CDP-DG is the source of the phosphatidyl moiety of PS, PE, and PC synthesized by the CDP-DG pathway as well as PI, phosphatidylglycerol, and cardiolipin (3, 4). CDP-choline and CDP-ethanolamine are the sources of the hydrophilic head groups of PC and PE synthesized by the Kennedy pathways, respectively (3, 4). Our laboratory utilizes the yeast Saccharomyces cerevisiae as a model eukaryote to study the regulation of CTP synthetase and its impact on phospholipid metabolism. CTP synthetase is encoded by ...

The pathogenic yeast, Candida albicans, is insensitive to the anti-mitotic drug, benomyl, and to the dihydrofolate reductase inhibitor, methotrexate. Genes responsible for the intrinsic drug resistance were sought by transforming Saccharomyces cerevisiae, a yeast sensitive to both drugs, with genomic C. albicans libraries and screening on benomyl or methotrexate. Restriction analysis of plasmids isolated from benomyl- and methotrexate-resistant colonies indicated that both phenotypes were encoded by the same DNA fragment. Sequence analysis showed that the fragments were nearly identical and contained a long open reading frame of 1694 bp (ORF1) and a small ORF of 446 bp (ORF2) within ORF1 on the opposite strand. By site-directed mutagenesis, it was shown that ORF1 encoded both phenotypes. The protein had no sequence similarity to any known proteins, including beta-tubulin, dihydrofolate reductase, and the P-glycoprotein of the multi-drug resistance family. The resistance gene was detected in several C. albicans strains and in C. stellatoidea by DNA hybridization and by the polymerase chain reaction.

In earlier studies we identified in a human genomic library a gene (human relaxin gene HI) coding for a relaxin-related peptide. We now have evidence that the human genome possesses an additional relaxin-related gene (designated human relaxin gene H2) which appears to be selectively expressed in the ovary during pregnancy. Nucleotide sequence analysis revealed striking differences in the predicted structures of relaxin encoded by these two genes. Chemical synthesis of biologically active relaxin based on the sequence obtained from ovarian cDNA clones confirmed that the expressed gene (H2) encodes an authentic human relaxin. The expressed gene appears to be transcribed into two different sized mRNAs and preliminary evidence suggests that the mRNA transcripts possess different 3'-untranslated regions. There was no evidence for the expression of human relaxin gene HI in the ovary and so far it is unclear whether gene Hl is expressed in another tissue or whether it represents a pseudogene. From the sequence data presented here it will now be possible to construct oligonucleotide probes and raise antibodies against synthetic peptides which could then be used to identify sites of relaxin biosynthesis and specifically quantitate the expression from either the Hl or H2 relaxin genes.