P-type ATPase from the cyanobacterium Synechococcus 7942 related to the human Menkes and Wilson disease gene products.

DNA encoding a P-type ATPase was cloned from the cyanobacterium Synechococcus 7942. The cloned ctaA gene encodes a 790-amino acid polypeptide related to the CopA Cu(2+)-uptake ATPase of Enterococcus hirae, to other known P-type ATPases, and to the candidate gene products for the human diseases of copper metabolism, Menkes disease and Wilson disease. Disruption of the single chromosomal gene in Synechococcus 7942 by insertion of an antibiotic-resistance cassette results in a mutant cell line with increased tolerance to Cu2+ compared with the wild type.     

NIP1, a gene required for nuclear transport in yeast.

Cytochrome c with a nuclear localization signal added at the N terminus was mistargeted to the nucleus, resulting in a yeast strain deficient in mitochondrial cytochrome c. Reversion of this strain allowed the isolation of temperature-conditional mutants defective in nuclear transport, as demonstrated with one of these mutants, nip1-1, that was shown to be defective in nuclear accumulation of a LacZ protein containing a nuclear localization signal of the yeast ribosomal protein L29. The NIP1+ gene was cloned and shown to encode a 93,143-Da protein. Furthermore, an epitope-labeled NIP1 protein migrated in SDS/polyacrylamide gels with a mass of approximately 100,000 Da and was shown by immunofluorescence to localize mainly in the cytoplasm. NIP1+ was shown to be an essential gene by gene disruption experiments. Intriguingly, NIP1 has a serine-rich acidic N-terminal region that is similar in this regard to the N-terminal region of a previously described nuclear localization signal-binding protein, NSR1.     

The canine copper toxicosis gene MURR1 is not implicated in the pathogenesis of Wilson disease

Background It has recently been demonstrated that the Wilson disease (WD) protein directly interacts with the human homolog of the MURR1 protein in vitro and in vivo, and that this interaction is specific for the copper transporter. The aim of the present study was to clarify the role of MURR1 in the pathogenesis of WD as well as in other WD-like disorders of hepatic copper metabolism of unknown origin. Methods Using the single-strand conformation polymorphism (SSCP) method followed by sequencing, we analyzed the 5′ untranslated region (UTR) and three exons of the MURR1 gene in three groups of patients: 19 wd patients in whom no mutations were detected in the ATP7B gene, 53 wd patients in whom only one mutation in the ATP7B gene was found, and 34 patients in whom clinical and laboratory data suggested a WD-like disorder of hepatic copper metabolism of unknown origin. Results We  detected in these patients six rare nucleotide substitutions, namely one splice-site consensus sequence and one missense and four silent nucleotide substitutions. All substitutions except one were found in the heterozygous state. No difference in the frequencies of the various substitutions was observed between patients and controls. Conclusions These data suggest that the MURR1 gene and its protein product are unlikely to play a primary role in the pathogenesis of Wilson disease. More extensive studies with larger numbers of clinically homogeneous patients should be carried out to establish whether nucleotide alterations in the MURR1 gene may have a role in causing WD or WD-like disorders or act as modifying factors in the phenotype variability in WD.     

A yeast gene required for DNA replication encodes a protein with homology to DNA helicases.

A yeast gene has been identified by screening for DNA replication mutants using a permeabilized cell replication assay. The mutant is temperature sensitive for growth and shows a cell cycle phenotype typical of DNA replication mutants. RNA synthesis is normal in the mutant but DNA synthesis ceases upon shift to the nonpermissive temperature. The DNA2 gene was cloned by complementation of the dna2ts gene phenotype. The gene is essential for viability. The gene encodes a 172-kDa protein with characteristic DNA helicase motifs. A hemagglutinin epitope-Dna2 fusion protein was prepared and purified by conventional and immunoaffinity chromatography. The purified protein is a DNA-dependent ATPase and has 3' to 5' DNA helicase activity specific for forked substrates. A nuclease activity that endonucleolytically cleaves DNA molecules having a single-stranded 5' tail adjacent to a duplex region copurifies through all steps with the fusion protein.     

Near-infrared fluorescent sensor for in vivo copper imaging in a murine Wilson disease model

Copper is an essential metal nutrient that is tightly regulated in the body because loss of its homeostasis is connected to severe diseases such as Menkes and Wilson diseases, Alzheimer's disease, prion disorders, and amyotrophic lateral sclerosis. The complex relationships between copper status and various stages of health and disease remain challenging to elucidate, in part due to a lack of methods for monitoring dynamic changes in copper pools in whole living organisms. Here we present the synthesis, spectroscopy, and in vivo imaging applications of Coppersensor 790, a first-generation fluorescent sensor for visualizing labile copper pools in living animals. Coppersensor 790 combines a near-infrared emitting cyanine dye with a sulfur-rich receptor to provide a selective and sensitive turn-on response to copper. This probe is capable of monitoring fluctuations in exchangeable copper stores in living cells and mice under basal conditions, as well as in situations of copper overload or deficiency. Moreover, we demonstrate the utility of this unique chemical tool to detect aberrant increases in labile copper levels in a murine model of Wilson disease, a genetic disorder that is characterized by accumulation of excess copper. The ability to monitor real-time copper fluxes in living animals offers potentially rich opportunities to examine copper physiology in health and disease.     

Tandem gene amplification mediates copper resistance in yeast.

Resistance to copper's toxicity in yeast is controlled by the CUP1r locus. This gene was cloned by transforming sensitive recipients (cup1(8)) with a collection of hybrid DNA molecules, consisting of random yeast DNA fragments inserted into the vector YRp7. Four resistant transformants were studied in detail. Autonomously replicating or integrated by homologous recombination into chromosomal sites, the corresponding plasmids and several subclones confer resistance on sensitive recipients carrying the natural variant allele, cup1(8). Tetrad analysis and genetic mapping established that integration occurs typically at the cup1(8) site located 28 centimorgans distal to thr1, a chromosome VIII marker. Restriction endonuclease cleavage and electrophoretic mobility studies revealed that the CUP1r locus consists of a tandem array of repetitive units. Each unit is 1.95 kilobases in length and contains single sites for Kpn I and Xba I and two Sau3A sites. The sensitive allele represents one repeat and the resistant allele embraces 15 tandemly arrayed repeat units. Progressive selections in higher copper concentrations establish strains with markedly enhanced resistance. Resistance, we propose, is mediated by a gene amplification mechanism based on unequal sister chromatid exchange.     

Characterization of VPS41, a gene required for vacuolar trafficking and high-affinity iron transport in yeast

Mutations in the yeast gene VPS41 give rise to poor growth on low iron medium, severe alterations in vacuolar morphology, and cause the missorting of membranous and soluble vacuolar proteins. Our studies predict that VPS41 encodes a hydrophilic protein of 992 amino acids that contains no obvious signal sequence or hydrophobic domains. The deduced Vps41p sequence contains a domain rich in glutamic and aspartic residues, as well as a domain with resemblance to a region of clathrin heavy chain. We have also identified and sequenced putative VPS41 homologues from Caenorhabditis elegans, plants, and humans. The VPS41 homologues (but not the yeast VPS41 itself) contain a conserved cysteine-rich RING-H2 zinc finger at their COOH termini. Biochemical experiments suggest that VPS41 functions in post-Golgi protein processing: the deletion mutant exhibits defective high affinity transport due to impaired Fet3p activity and also exhibits defects in the processing and sorting of multiple vacuolar hydrolases.     

Truncating mutations in the Wilson disease gene ATP7B are associated with very low serum ceruloplasmin oxidase activity and an early onset of Wilson disease

Background  

Mutations in the gene ATP7B cause Wilson disease, a copper storage disorder with a high phenotypic and genetic heterogeneity. We aimed to evaluate whether 'severe' protein-truncating ATP7B mutations (SMs) are associated with low serum ceruloplasmin oxidase activities and an early age of onset when compared to missense mutations (MMs).     

Cloning and characterization of a cellular apoptosis susceptibility gene, the human homologue to the yeast chromosome segregation gene CSE1.

We recently isolated human cDNA fragments that render MCF-7 breast cancer cells resistant to cell death caused by Pseudomonas exotoxin, Pseudomonas exotoxin-derived immunotoxins, diphtheria toxin, and tumor necrosis factor. We report here that one of these fragments is an antisense fragment of a gene homologous to the essential yeast chromosome segregation gene CSE1. Cloning and analysis of the full-length cDNA of the human CSE1 homologue, which we name CAS for cellular apoptosis susceptibility gene, reveals a protein coding region with similar length (971 amino acids for CAS, 960 amino acids for CSE1) and 59% overall protein homology to the yeast CSE1 protein. The conservation of this gene indicates it has an important function in human cells consistent with the essential role of CSE1 in yeast. CAS is highly expressed in human tumor cell lines and in human testis and fetal liver, tissues that contain actively dividing cells. Furthermore, CAS expression increases when resting human fibroblasts are induced to proliferate and decreases when they are growth-arrested. Thus, CAS appears to play an important role in both toxin and tumor necrosis factor-mediated cell death, as well as in cell proliferation.     

The yeast nuclear import receptor is required for mitosis.

The nuclear import system is highly conserved among eukaryotes. Here we report the effects of a conditional mutation in SRP1, which encodes a Saccharomyces cerevisiae homolog of the vertebrate nuclear import receptor importin. Importin was isolated as a factor required for the initial targeting step of a nuclear import substrate to the nuclear envelope in a mammalian in vitro assay. We show that yeast Srp1 is similarly required for protein import. In addition, Srp1 is also required for the execution of mitosis: we demonstrate that cells containing a conditional mutation of SRP1 arrest with a G2/M phenotype in a manner analogous to classic cdc mutants. This defect may be due to the failure of the mutant to degrade the mitotic cyclin Clb2 and other proteins required for mitosis. The requirement of a nuclear import receptor for cell cycle-regulated proteolysis implies that import of cell cycle regulators into the nucleus is critical for cell cycle progression.     

hCTR1: A human gene for copper uptake identified by complementation in yeast

The molecular mechanisms responsible for the cellular uptake of copper in mammalian cells are unknown. We describe isolation of a human gene involved in this process by complementation of the yeast high-affinity copper uptake mutant, ctr1. Besides complementing ctr1 growth defect on nonfermentable media, the human gene also rescues iron transport and SOD1 defects in ctr1 yeast. Overexpression of the gene in yeast leads to vulnerability to the toxicity of copper overload. In addition, its expression in ctr1 yeast significantly increases the level of cellular copper, as demonstrated by atomic absorption. We propose this gene as a candidate for high-affinity copper uptake in humans and by analogy have named it hCTR1. The hCTR1 and yeast CTR1 predicted transmembrane proteins are 29% identical, but the human protein is substantially smaller in both the extracellular metal-binding and intracellular domains. An additional human gene similar to hCTR1, here named hCTR2, was identified in a database search. Both hCTR1 and hCTR2 are expressed in all human tissues examined, and both genes are located in 9q31/32. These studies, together with the previously recognized functional and sequence similarity between the Menkes/Wilson copper export proteins and CCC2 in yeast, demonstrate that similar copper homeostatic mechanisms are used in these evolutionarily divergent organisms.     

Assignment of the gene for Wilson disease to chromosome 13: linkage to the esterase D locus.

Wilson disease (WD) is an autosomal recessively inherited disorder of copper metabolism for which the basic defect is still unknown. Twenty-seven autosomal markers were investigated for linkage in a large inbred kindred with affected individuals in two generations. Also, serum copper and ceruloplasmin were measured on all available members. Close linkage (theta = 0.06) with a logarithm of odds (lod) score of 3.21 was found between the gene for WD and the esterase D locus. Efficient detection of linkage was made possible by the use of a multisibship inbred pedigree. The discovery of a polymorphic marker genetically linked to the WD locus has profound implications both for investigation of the primary gene defect and for clinical services.     

ELG1, a yeast gene required for genome stability,forms a complex related to replication factor C

Many overlapping surveillance and repair mechanisms operate in eukaryotic cells to ensure the stability of the genome. We have screened to isolate yeast mutants exhibiting increased levels of recombination between repeated sequences. Here we characterize one of these mutants, elg1. Strains lacking Elg1p exhibit elevated levels of recombination between homologous and nonhomologous chromosomes, as well as between sister chromatids and direct repeats. These strains also exhibit increased levels of chromosome loss. The Elg1 protein shares sequence homology with the large subunit of the clamp loader replication factor C (RFC) and with the product of two additional genes involved in checkpoint functions and genome maintenance: RAD24 and CTF18. Elg1p forms a complex with the Rfc2-5 subunits of RFC that is distinct from the previously described RFC-like complexes containing Rad24 and Ctf18. Genetic data indicate that the Elg1, Ctf18, and Rad24 RFC-like complexes work in three separate pathways important for maintaining the integrity of the genome and for coping with various genomic stresses.     

Fte-1, a v-fos transformation effector gene, encodes the mammalian homologue of a yeast gene involved in protein import into mitochondria.

Revertants were isolated from v-fos-transformed rat-1 cells cotransfected with a human cDNA expression library and a selectable marker (pMEX-neo). Molecular analysis of one clone, R2.2, suggested that the revertant phenotype resulted from the disruption of a transformation effector gene by the integration of the pMEX-neo plasmid. Genomic sequences flanking the plasmid integration site were cloned and used as probes in Northern blot analyses. A probe derived from sequences 5' to the integration site hybridized to a unique 1.2-kilobase mRNA and was used to isolate a 0.9-kilobase cDNA clone (fte-1). The open reading frame of the fte-1 cDNA predicts a highly basic protein that shows a remarkable level of similarity with two genes from Saccharomyces cerevisiae. One of these yeast genes contains an unidentified open reading frame and the other, MFT1, is a gene isolated from a yeast mutant that fails to import a fusion protein into mitochondria [Garrett, J. M., Singh, K. K., Vonder Haar, R. A. & Emr, S. D. (1991) Mol. Gen. Genet. 225, 483-491]. Expression of the fte-1 gene was induced approximately 5-fold in v-fos-transformed fibroblasts, but expression was reduced in clone R2.2 and in several independent revertant clones. Transfection of R2.2 cells with fte-1 expression vectors resulted in the reacquisition of a transformed phenotype. These results demonstrate that the mammalian homologue of a gene implicated in protein import into yeast mitochondria is a v-fos transformation effector gene.     

The RAD52 gene is required for homothallic interconversion of mating types and spontaneous mitotic recombination in yeast.

The rad52-1 mutation prevents homothallic mating type interconversion and reduces mitotic recombination in yeast. It has been previously reported that rad52-1 abolishes meiotic recombination. These data suggest either that a generalized recombination function(s) is required for mating type switching or that generalized recombination and specific homothallic functions are jointly controlled by the RAD52 gene. The rad52-1 mutation affects the interconversion of the two yeast mating types (a and alpha) differently, suggesting that the interconversion process is not equivalent for both mating types. This type of asymmetry is not predicted by current models of homothallic switching.