ABSTRACT
Single electron spins bound to multi-phosphorus nuclear spin registers in silicon have demonstrated fast (0.8 ns) two-qubit SWAP gates and long spin relaxation times (~30 s). In these spin registers, when the donors are ionized, the nuclear spins remain weakly coupled to their environment, allowing exceptionally long coherence times. When the electron is present, the hyperfine interaction allows coupling of the spin and charge degrees of freedom for fast qubit operation and control. Here we demonstrate the use of the hyperfine interaction to enact electric dipole spin resonance to realize high-fidelity ( F = 10 0 - 6 + 0 %) initialization of all the nuclear spins within a four-qubit nuclear spin register. By controllably initializing the nuclear spins to â â â , we achieve single-electron qubit gate fidelities of F = 99.78 ± 0.07% (Clifford gate fidelities of 99.58 ± 0.14%), above the fault-tolerant threshold for the surface code with a coherence time of T 2 * = 12 µ s .
ABSTRACT
The analysis of large-scale gene expression profiles is still a demanding and extensive task. Modern machine learning and data mining techniques developed in linear algebra, like Independent Component Analysis (ICA), become increasingly popular as appropriate tools for analyzing microarray data. We applied ICA to analyze kinetic gene expression profiles of human monocyte derived macrophages (MDM) from three different donors infected with Francisella tularensis holartica and compared them to more classical methods like hierarchical clustering. Results were compared using a pathway analysis tool, based on the Gene Ontology and the MeSH database. We could show that both methods lead to time-dependent gene regulatory patterns which fit well to known TNFalpha induced immune responses. In comparison, the nonexclusive attribute of ICA results in a more detailed view and a higher resolution in time dependent behavior of the immune response genes. Additionally, we identified NFkappaB as one of the main regulatory genes during response to F. tularensis infection.
Subject(s)
Francisella tularensis/physiology , Gene Expression Profiling/methods , Macrophages/physiology , Oligonucleotide Array Sequence Analysis/methods , Principal Component Analysis , Tularemia/genetics , Algorithms , Cells, Cultured , Cluster Analysis , Gene Regulatory Networks , Humans , Macrophages/metabolism , Macrophages/microbiology , Models, Genetic , Tularemia/metabolismABSTRACT
INTRODUCTION: Apolipoprotein A-IV (apoA-IV), an intestinally and cerebrally synthesized satiety factor and anti-atherogenic plasma apolipoprotein, was recently identified as an anti-inflammatory protein. In order to elucidate whether intestinal apoA-IV exerts similar repair function as its hepatic homologue apolipoprotein A-V (apoA-V), apoA-IV-interactive proteins were searched and in vitro functional studies were performed with apoA-IV overexpressing cells. ApoA-IV was also analyzed in the intestinal mucosa of patients with inflammatory bowel diseases (IBD), together with other genes involved in epithelial junctional integrity. METHODS: A yeast-two-hybrid screening was used to identify apoA-IV-interactors. ApoA-IV was overexpressed in Caco-2 and HT-29 mucosal cells for colocalization and in vitro epithelial permeability studies. Mucosal biopsies from quiescent regions of colon transversum and terminal ileum were subjected to DNA-microarray analysis and pathway-related data mining. RESULTS: Four proteins interacting with apoA-IV were identified, including apolipoprotein B-100, alpha1-antichymotrypsin, cyclin C, and the cytosolic adaptor alpha-catenin, thus linking apoA-IV to adherens junctions. Overexpression of apoA-IV was paralleled with a differentiated phenotype of intestinal epithelial cells, upregulation of junctional proteins, and decreased paracellular permeability. Colocalization between alpha-catenin and apoA-IV occurred exclusively in junctional complexes. ApoA-IV was downregulated in quiescent mucosal tissues from patients suffering from IBD. In parallel, only a distinct set of junctional genes was dysregulated in non-inflamed regions of IBD gut. CONCLUSIONS: ApoA-IV may act as a stabilizer of adherens junctions interacting with alpha-catenin, and is likely involved in the maintenance of junctional integrity. ApoA-IV expression is significantly impaired in IBD mucosa, even in non-inflamed regions.
Subject(s)
Apolipoproteins A/metabolism , Apolipoproteins A/physiology , Cell Membrane Permeability/genetics , Inflammatory Bowel Diseases/genetics , Inflammatory Bowel Diseases/metabolism , Intestinal Mucosa/physiology , alpha Catenin/metabolism , Apolipoproteins A/genetics , Caco-2 Cells , Gene Expression Profiling , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HT29 Cells , Humans , Inflammatory Bowel Diseases/pathology , Intercellular Junctions/metabolism , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Oligonucleotide Array Sequence Analysis , Protein Binding , RNA, Messenger/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Satiation/physiology , TransfectionABSTRACT
The yeast, Saccharyomyces cerevisiae, has become an important organism in molecular, biochemical, and genetic analysis. The organism has specific requirements for growth under a variety of conditions. The media, both liquid and solid, simple, define, and complex are describe in this unit. Also included are methods for handling, storing, and shipping stock of yeast.
Subject(s)
Culture Media/chemistry , Culture Media/pharmacology , Saccharomyces cerevisiae/drug effects , Cell Culture Techniques/methods , Cell Proliferation/drug effects , Culture Media/chemical synthesis , Drug Contamination/prevention & control , Saccharomyces cerevisiae/metabolism , Sterilization/methods , Sterilization/standards , TemperatureABSTRACT
Yeast and other fungi contain a soluble elongation factor 3 (EF-3) which is required for growth and protein synthesis. EF-3 contains two ABC cassettes, and binds and hydrolyses ATP. We identified a homolog of the YEF3 gene in the Saccharomyces cerevisiae genome database. This gene, designated YEF3B, is 84% identical in protein sequence to YEF3, which we will now refer to as YEF3A. YEF3B is not expressed during growth under laboratory conditions, and thus cannot rescue growth of YEF3A deletion strains. However, YEF3B can take the place of YEF3A in vivo when expressed from the YEF3A or ADH1 promoters. The products of the YEF3A and YEF3B genes, EF-3A and EF-3B, respectively, were expressed from the ADH1 promoter and purified. Both factors possessed basal and ribosomal-stimulated ATPase activity, and had similar affinity for yeast ribosomes (103 to 113 nM). K(m) values for ATP were similar, but the Kcat values differed significantly. Ribosome-dependent ATPase activity of EF-3A was more efficient than EF-3B, since the Kcat and Kcat/K(m) values for EF-3A were about two-fold higher; however, the difference in Kcat/K(m) values between the two factors was small for basal ATPase activity.
Subject(s)
Genes, Fungal , Peptide Elongation Factors/chemistry , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Base Sequence , Blotting, Northern , Blotting, Western , Gene Expression Regulation, Fungal/genetics , Gene Expression Regulation, Fungal/physiology , Molecular Sequence Data , Peptide Elongation Factors/metabolism , Polymerase Chain ReactionABSTRACT
We report the identification and characterization of CTR1, a gene in the yeast S. cerevisiae that encodes a multispanning plasma membrane protein specifically required for high affinity copper transport into the cell. The predicted protein contains a methionine- and serine-rich domain that includes 11 examples of the sequence Met-X2-Met, a motif noted in proteins involved in bacterial copper metabolism. CTR1 mutants and deletion strains have profound deficiency in ferrous iron uptake, thus revealing a requirement for copper in mediating ferrous transport into the cell. Genetic evidence suggests that the target for this requirement is the FET3 gene (detailed in a companion study), predicted to encode a copper-containing protein that acts as a cytosolic ferro-oxidase. These findings provide an unexpected mechanistic link between the uptake of copper and iron.
Subject(s)
Cation Transport Proteins , Copper/metabolism , Fungal Proteins/genetics , Genes, Fungal , Membrane Proteins/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Base Sequence , Biological Transport , Cloning, Molecular , Copper Transporter 1 , Ferrous Compounds/metabolism , Fungal Proteins/metabolism , Iron/metabolism , Membrane Proteins/metabolism , Molecular Sequence Data , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
An amino acid limitation in bacteria elicits a global response, called stringent control, that leads to reduced synthesis of rRNA and ribosomal proteins and increased expression of amino acid biosynthetic operons. We have used the antimetabolite 3-amino-1,2,4-triazole to cause histidine limitation as a means to elicit the stringent response in the yeast Saccharomyces cerevisiae. Fusions of the yeast ribosomal protein genes RPL16A, CRY1, RPS16A, and RPL25 with the Escherichia coli lacZ gene were used to show that the expression of these genes is reduced by a factor of 2 to 5 during histidine-limited exponential growth and that this regulation occurs at the level of transcription. Stringent regulation of the four yeast ribosomal protein genes was shown to be associated with a nucleotide sequence, known as the UASrpg (upstream activating sequence for ribosomal protein genes), that binds the transcriptional regulatory protein RAP1. The RAP1 binding sites also appeared to mediate the greater ribosomal protein gene expression observed in cells growing exponentially than in cells in stationary phase. Although expression of the ribosomal protein genes was reduced in response to histidine limitation, the level of RAP1 DNA-binding activity in cell extracts was unaffected. Yeast strains bearing a mutation in any one of the genes GCN1 to GCN4 are defective in derepression of amino acid biosynthetic genes in 10 different pathways under conditions of histidine limitation. These Gcn- mutants showed wild-type regulation of ribosomal protein gene expression, which suggests that separate regulatory pathways exist in S. cerevisiae for the derepression of amino acid biosynthetic genes and the repression of ribosomal protein genes in response to amino acid starvation.
Subject(s)
DNA, Fungal/genetics , DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Genes, Fungal , Ribosomal Proteins/genetics , Saccharomyces cerevisiae/genetics , Transcription Factors , Transcription, Genetic , Amitrole/pharmacology , Base Sequence , Binding Sites , Cloning, Molecular , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/metabolism , Genotype , Histidine/metabolism , Histidine/pharmacology , Kinetics , Molecular Sequence Data , Recombinant Fusion Proteins/biosynthesis , Ribosomal Proteins/biosynthesis , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolismABSTRACT
Glucose represses PRB1 expression at the level of transcription. However, release from glucose repression initially does not result in accumulation of protease B (PrB) activity despite transcriptional derepression. PrB activity accumulates only upon a second transcriptional derepression as the cells approach stationary phase. Increasing the PRB1 gene dosage on 2 mu-based plasmids does not overcome glucose repression. Glucose-mediated repression of PRB1 is not subject to the same genetic controls as SUC2. Mutation of the HXK2 gene, which confers glucose-insensitive expression of secreted invertase, had no effect on PRB1 expression at the level of PrB activity. Strains bearing a mutation in any of the SNF1-SNF6 genes cannot derepress secreted invertase synthesis, but did derepress PrB synthesis when grown in the absence of glucose. Mutation of the SNF2 or SNF5 gene led to accumulation of PrB activity to levels ten times that of wild type. Polymorphism for a suppressor gene was observed: in snf5-bearing strains, one allele of this suppressor gene resulted in elevated levels of PrB and the other allele resulted in wild-type levels of PrB; neither allele suppressed the Suc- phenotype of the snf5 mutant. Re-examination of published data on SUC2 expression in snf2 and snf5 mutants and examination of PRB1 expression in these mutants paradoxically suggest that the SNF2 and SNF5 gene products might act as negative regulators of gene expression.
Subject(s)
Gene Expression Regulation, Fungal , Genes, Fungal , Glucose/pharmacology , Mutation , Saccharomyces cerevisiae/genetics , Serine Endopeptidases/genetics , Culture Media , Dosage Compensation, Genetic , Gene Expression Regulation, Enzymologic , Kinetics , Phenotype , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/growth & development , Serine Endopeptidases/metabolism , Transcription, GeneticABSTRACT
The vacuolar protease B of Saccharomyces cerevisiae is a subtilisin-like protease encoded by the PRB1 gene. Antibodies raised against a synthetic peptide and an Escherichia coli-derived PRB1 open reading frame (ORF) protein cross-react with authentic protease B from yeast. By using these antibodies, the posttranslational biosynthetic pathway of protease B has been elucidated. Preproprotease B is a 76-kD unglycosylated precursor that enters the endoplasmic reticulum (ER), where it receives one asparagine-linked (Asn-linked) and an undetermined number of non-Asn-linked carbohydrate side chains. The large glycosylated intermediate is proteolytically processed to a 39-kD form before exiting the ER. In the Golgi complex, the 39-kD form becomes 40 kD, due to elaboration of the Asn-linked side chain. The carboxyterminal end of the 40-kD proprotease B undergoes protease A-mediated processing to a 37-kD intermediate, which in turn is quickly processed to 31-kD mature protease B. The ultimate processing step removes a peptide containing the Asn-linked chain; mature PrB has only non-Asn-linked carbohydrate.
Subject(s)
Saccharomyces cerevisiae/enzymology , Serine Endopeptidases/biosynthesis , Asparagine/metabolism , Cytoplasm/enzymology , Endoplasmic Reticulum/enzymology , Enzyme Precursors/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Glycosylation , Golgi Apparatus/enzymology , Immunoblotting , Immunosorbent Techniques , Mutation , Protein Processing, Post-Translational , Saccharomyces cerevisiae/genetics , Serine Endopeptidases/genetics , Vacuoles/enzymologyABSTRACT
The PRB1 gene of Saccharomyces cerevisiae encodes the vacuolar endoprotease protease B. We have determined the DNA sequence of the PRB1 gene and the amino acid sequence of the amino terminus of mature protease B. The deduced amino acid sequence of this serine protease shares extensive homology with those of subtilisin, proteinase K, and related proteases. The open reading frame of PRB1 consists of 635 codons and, therefore, encodes a very large protein (molecular weight, greater than 69,000) relative to the observed size of mature protease B (molecular weight, 33,000). Examination of the gene sequence, the determined amino-terminal sequence, and empirical molecular weight determinations suggests that the preproenzyme must be processed at both amino and carboxy termini and that asparagine-linked glycosylation occurs at an unusual tripeptide acceptor sequence.
Subject(s)
Base Sequence , DNA, Fungal/genetics , Lysosomes/enzymology , Organoids/enzymology , Saccharomyces cerevisiae/enzymology , Sequence Homology, Nucleic Acid , Serine Endopeptidases/genetics , Subtilisins/genetics , Vacuoles/enzymology , Amino Acid Sequence , Codon , Computers , Electrophoresis, Polyacrylamide Gel , Endopeptidase K , Molecular Sequence Data , Molecular Weight , Saccharomyces cerevisiae/geneticsABSTRACT
We have isolated the structural gene, PRB1, for the vacuolar protease B of Saccharomyces cerevisiae from a genomic library by complementation of the prb1-1122 mutation. Deletion analysis localized the complementing activity to a 3.2-kilobase pair XhoI-HindIII restriction enzyme fragment. The fragment was used to identify a 2.3-kilobase mRNA. S1 endonuclease mapping indicated that the mRNA and the gene were colinear. No introns were detected. The mRNA is of sufficient size to encode a protein of about 69,000 molecular weight, a number much larger than either the mature enzyme (congruent to 30,000 protein molecular weight) or the sole reported precursor (congruent to 39,000 protein molecular weight). These results suggest that proteolytic processing steps beyond that thought to be catalyzed by protease A may be required to convert the initial glycosylated translation product into mature protease B. The PRB1 mRNA is made in substantial amounts only when the cells have exhausted the glucose supply and enter the diauxic plateau. There is an extended time lag between PRB1 transcription and expression of protease B activity. A deletion that removes about 83% of the coding region was constructed as a diploid heterozygote. Spores bearing the deletion germinate, grow at normal rates into colonies, and have no obvious phenotype beyond protease B deficiency.
Subject(s)
Endopeptidases/genetics , Genes, Fungal , Genes , Saccharomyces cerevisiae/genetics , Serine Endopeptidases , Genetic Complementation Test , Mutation , Nucleic Acid Hybridization , RNA, Messenger/genetics , Saccharomyces cerevisiae/enzymology , Vacuoles/enzymologyABSTRACT
It is well established that the administration to rodents of a variety of structurally diverse chemicals possessing hypotriglyceridemic properties results in hepatomegaly, the induction of hepatic peroxisome (microbody) proliferation, and the development of hepatocellular carcinomas. Studies have led to the hypothesis that persistent proliferation of peroxisomes serves as an endogenous initiator of neoplastic transformation in liver by increasing the intracellular production of H2O2 by the peroxisomal oxidase(s). The objective of the present study was to determine whether hepatic peroxisome proliferation can be induced in cats, chickens, pigeons, and two species of monkeys (rhesus and cynomolgus). The hypolipidemic drug ciprofibrate (2-[4-(2,2-dichloro-cylopropyl)phenoxyl]2-methylpropionic acid) induced peroxisome proliferation in the livers of cats (dose, greater than 40 mg/kg body weight for 4 weeks); chickens (dose greater than 25 mg/kg body weight for 4 weeks); pigeons (300 mg/kg body weight for 3 weeks), rhesus monkeys (50 to 200 mg/kg body weight for 7 weeks) and cynomolgus monkeys (400 mg/kg body weight for 4 weeks). In all five species examined in this study, a marked but variable increase in the activities of peroxisomal catalase, carnitine acetyltransferase, heat-labile enoyl-CoA hydratase, and the fatty acid beta-oxidation system was observed. These results suggest that peroxisome proliferation can be induced in the livers of several species and that it is a dose-dependent but not a species-specific phenomenon.