Redundant 3' end-forming signals for the yeast CYC1 mRNA.

The cyc1-512 mutation is a 38-bp deletion in the 3' untranslated region of the CYC1 gene, which encodes iso-1-cytochrome c in Saccharomyces cerevisiae. This deletion caused a 90% reduction in the levels of the CYC1 mRNA and protein because of the absence of the normal 3' end-forming signal. Although the 3' end-forming signal was not defined by previous analyses, we report that concomitant alteration by base-pair substitution of three 3' end-forming signals within and adjacent to the 38-bp region produced the same phenotype as the cyc1-512 mutation. Furthermore, these signals appear to be related to the previously identified 3' end-forming signal TATATA. A computer analysis revealed that TATATA and related sequences were present in the majority of 3' untranslated regions of yeast genes. Although TATATA may be the strongest and most frequently used signal in yeast genes, the CYC1+ gene concomitantly employed the weaker signals TT-TATA, TATGTT, and TATTTA, resulting in a strong signal.

Escherichia coli iron superoxide dismutase targeted to the mitochondria of yeast cells protects the cells against oxidative stress.

A gene encoding a fusion protein consisting of Escherichia coli iron superoxide dismutase (FeSOD) with the mitochondrial targeting presequence of yeast manganese superoxide dismutase (MnSOD) was cloned and expressed in E. coli and in Saccharomyces cerevisiae DL1Mn- yeast cells deficient in MnSOD. In the yeast cells the fusion protein was imported into the mitochondrial matrix. However, the presequence was not cleaved. In a control set of experiments, the E. coli FeSOD gene without the yeast MnSOD leader sequence was also cloned and expressed in S. cerevisiae DL1Mn- cells. In this case the FeSOD was located in the cytosol and was not imported into the mitochondrial matrix. E. coli FeSOD, with and without the yeast MnSOD presequence, proved to be active in yeast, but, whereas the FeSOD targeted to the mitochondria of yeast cells deficient in MnSOD protected the cells from the toxic effects of oxidative stress, FeSOD without the yeast MnSOD presequence did not protect the yeast cells deficient in MnSOD against oxidative stress.

Analysis of perinatal gene expression: hormone response elements mediate activation of a lacZ reporter gene in liver of transgenic mice.

The transcription of genes encoding gluconeogenic enzymes is tightly regulated during the perinatal period. These genes are induced by glucagon (cAMP) and glucocorticoids and repressed by insulin. To address the role of cAMP and glucocorticoids in the physiological activation of genes encoding gluconeogenic enzymes in the perinatal period, transgenic mice have been generated with chimeric constructs containing the reporter gene lacZ under the control of hormone response elements. The activity of the transgene is restricted to the liver by the presence of the enhancers from the alpha-fetoprotein gene and its transcription is driven by a promoter that contains a TATA box linked to either cAMP response elements (CREs) or glucocorticoid response elements (GREs). We demonstrate cAMP and glucocorticoid regulation, liver-specific expression, and perinatal activation of the reporter gene. These data indicate that the CRE and GRE are, independently, necessary and sufficient to mediate perinatal gene activation. Perinatal activation was not impaired when a CRE reporter transgene was assayed in mice that contain a targeted mutation of the CRE-binding protein (CREB) gene, providing further evidence for functional redundancy among the members of the CREB/ATF gene family.

Diminished degradation of yeast cytochrome c by interactions with its physiological partners.

The level and structure of yeast iso-1-cytochrome c and iso-2-cytochrome c, encoded by the nuclear genes CYC1 and CYC7, respectively, are normally not altered in rho- mutants, which completely lack the cytochromes a.a3 subunits and cytochrome b that are encoded by mitochondrial DNA. In contrast, iso-cytochromes c containing the amino acid change Thr-78-->Ile (T78I) were observed at the normal or near-normal wild-type level in rho+ strains but were completely absent in rho- mutants. We have demonstrated with the "global" suppressor mutation Asn-52-->Ile and by pulse-chase labeling that the T78I iso-1-cytochrome c undergoes rapid cellular degradation in rho- mutants. Furthermore, specific mutations revealed that the deficiency of T78I iso-1 cytochrome c can be caused by the lack of cytochrome a.a3 or cytochrome c1, but not by the lack of cytochrome b. Thus, this and certain other, but not all, labile forms of cytochrome c are protected from degradation by the interaction with its physiological partners.

Cyclin-dependent protein kinase and cyclin homologs SSN3 and SSN8 contribute to transcriptional control in yeast.

The SSN3 and SSN8 genes of Saccharomyces cerevisiae were identified by mutations that suppress a defect in SNF1, a protein kinase required for release from glucose repression. Mutations in SSN3 and SSN8 also act synergistically with a mutation of the MIG1 repressor protein to relieve glucose repression. We have cloned the SSN3 and SSN8 genes. SSN3 encodes a cyclin-dependent protein kinase (cdk) homolog and is identical to UME5. SSN8 encodes a cyclin homolog 35% identical to human cyclin C. SSN3 and SSN8 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. Using an immune complex assay, we detected protein kinase activity that depends on both SSN3 and SSN8. Thus, the two SSN proteins are likely to function as a cdk-cyclin pair. Genetic analysis indicates that the SSN3-SSN8 complex contributes to transcriptional repression of diversely regulated genes and also affects induction of the GAL1 promoter.

Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast.

The CDC47 gene was isolated by complementation of a cdc47 temperature-sensitive mutant in Saccharomyces cerevisiae and was shown to encode a predicted polypeptide, Cdc47, of 845 aa. Cdc47 belongs to the Cdc46/Mcm family of proteins, previously shown to be essential for initiation of DNA replication. Using indirect immunofluorescence microscopy and subcellular fractionation techniques, we show that Cdc47 undergoes cell cycle-regulated changes in its subcellular localization. At mitosis, Cdc47 enters the nucleus, where it remains until soon after the initiation of DNA replication, when it is rapidly exported back into the cytoplasm. Cdc47 protein levels do not vary with the cell cycle, but expression of CDC47 and nascent synthesis of Cdc47 occur late in the cell cycle, coinciding with mitosis. Together, these results show that Cdc47 is not only imported into the nucleus at the end of mitosis but is also exported back into the cytoplasm at the beginning of S phase. The observation that Cdc47 is exported from the nucleus at the beginning of S phase has important implications for how initiation of DNA replication is controlled.

The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake.

The CCC2 gene of the yeast Saccharomyces cerevisiae is homologous to the human genes defective in Wilson disease and Menkes disease. A biochemical hallmark of these diseases is a deficiency of copper in ceruloplasmin and other copper proteins found in extracytosolic compartments. Here we demonstrate that disruption of the yeast CCC2 gene results in defects in respiration and iron uptake. These defects could be reversed by supplementing cells with copper, suggesting that CCC2 mutant cells were copper deficient. However, cytosolic copper levels and copper uptake were normal. Instead, CCC2 mutant cells lacked a copper-dependent oxidase activity associated with the extracytosolic domain of the FET3-encoded protein, a ceruloplasmin homologue previously shown to be necessary for high-affinity iron uptake in yeast. Copper restored oxidase activity both in vitro and in vivo, paralleling the ability of copper to restore respiration and iron uptake. These results suggest that the CCC2-encoded protein is required for the export of copper from the cytosol into an extracytosolic compartment, supporting the proposal that intracellular copper transport is impaired in Wilson disease and Menkes disease.