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PubMed1996

A phosphoglucomutase-like gene essential for the optimal expression of methicillin resistance in Staphylococcus aureus: molecular cloning and DNA sequencing.

We describe here the cloning and sequencing of a new auxiliary gene identified by Tn551 insertional mutagenesis of the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL. The insertionally inactivated mutant RUSA315 had intact mecA and normal amounts of PBP2A, but drastically reduced antibiotic resistance (drop in methicillin MIC from 1600 to 1.5 micrograms ml-1), a unique heterogeneous phenotype, and a compositional change in the cell wall characterized by the complete disappearance of the unsubstituted disaccharide pentapeptide from the peptidoglycan. Cloning in E. coli followed by sequencing located the Tn551 insert omega 720 in an open reading frame of 451 codons, provisionally called femR315, defining a polypeptide with a deduced amino acid sequence that showed over 26% sequence identity and 57% overall sequence similarity with the phosphoglucomutase (PGM) gene of E. coli. The Tn551 insertion site of a previously described mutant 12F (femD) also lies in the same gene as femR315. The wild-type form of femR315 subcloned in a shuttle vector fully restored expression of high level (parental) methicillin resistance in mutant RUSA315. The exact biochemical function of femR315 is not known. However, enzymes similar to PGM catalyze the isomerization of hexose and hexosamine phosphates leading to the formation of glucosamine-1-P, which is an obligate precursor in the biosynthesis of UDP-N-acetylglucosamine (UDP-NAGA). We propose that the suppression of methicillin resistance in RUSA315 is related to some functional or quantitative abnormality of UDP-NAGA metabolism.

Amino Acid Sequence↗
PubMed2003

Identification and characterization of Helicobacter pylori genes essential for gastric colonization.

Helicobacter pylori causes one of the most common, chronic bacterial infections and is a primary cause of severe gastric disorders. To unravel the bacterial factors necessary for the process of gastric colonization and pathogenesis, signature tagged mutagenesis (STM) was adapted to H. pylori. The Mongolian gerbil (Meriones unguiculatus) was used as model system to screen a set of 960 STM mutants. This resulted in 47 H. pylori genes, assigned to 9 different functional categories, representing a set of biological functions absolutely essential for gastric colonization, as verified and quantified for many mutants by competition experiments. Identification of previously known colonization factors, such as the urease and motility functions validated this method, but also novel and several hypothetical genes were found. Interestingly, a secreted collagenase, encoded by hp0169, could be identified and functionally verified as a new essential virulence factor for H. pylori stomach colonization. Furthermore, comB4, encoding a putative ATPase being part of a DNA transformation-associated type IV transport system of H. pylori was found to be absolutely essential for colonization, but natural transformation competence was apparently not the essential function. Thus, this first systematic STM application identified a set of previously unknown H. pylori colonization factors and may help to potentiate the development of novel therapies against gastric Helicobacter infections.

Animals↗
PubMed1995

Yeast mRNA cap methyltransferase is a 50-kilodalton protein encoded by an essential gene.

RNA (guanine-7-)methyltransferase, the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA, was isolated from extracts of Saccharomyces cerevisiae. The yeast enzyme catalyzed methyl group transfer from S-adenosyl-L-methionine to the guanosine base of capped, unmethylated poly(A). Cap methylation was stimulated by low concentrations of salt and was inhibited by S-adenosyl-L-homocysteine, a presumptive product of the reaction, but not by S-adenosyl-D-homocysteine. The methyltransferase sedimented in a glycerol gradient as a single discrete component of 3.2S. A likely candidate for the gene encoding yeast cap methyltransferase was singled out on phylogenetic grounds. The ABD1 gene, located on yeast chromosome II, encodes a 436-amino-acid (50-kDa) polypeptide that displays regional similarity to the catalytic domain of the vaccinia virus cap methyltransferase. That the ABD1 gene product is indeed RNA (guanine-7-)methyltransferase was established by expressing the ABD1 protein in bacteria, purifying the protein to homogeneity, and characterizing the cap methyltransferase activity intrinsic to recombinant ABD1. The physical and biochemical properties of recombinant ABD1 methyltransferase were indistinguishable from those of the cap methyltransferase isolated and partially purified from whole-cell yeast extracts. Our finding that the ABD1 gene is required for yeast growth provides the first genetic evidence that a cap methyltransferase (and, by inference, the cap methyl group) plays an essential role in cellular function in vivo.

Amino Acid Sequence↗
PubMed2001

Isolation and characterization of acetoacetyl-CoA thiolase gene essential for n-decane assimilation in yeast Yarrowia lipolytica.

Yarrowia lipolytica is a yeast which can utilize n-alkane as a sole carbon source. We isolated a Y. lipolytica peroxisomal acetoacetyl-CoA thiolase gene, PAT1, by complementation of a mutant that cannot utilize n-decane as a sole carbon source. We found that the putative PAT1 product had conserved features of peroxisomal acetoacetyl-CoA thiolase. We showed that the PAT1 disruptant was not able to grow on n-decane, and that n-decane-inducible acetoacetyl-CoA thiolase activity largely depended on PAT1. The original mutant carried a mutation involving the replacement of Gly382 with Glu. This mutation inactivated the ability of PAT1 to complement the defective n-decane utilization of the disruptant. These results indicate that PAT1 encodes peroxisomal acetoacetyl-CoA thiolase and is essential for n-decane utilization in Y. lipolytica.

Acetyl-CoA C-Acetyltransferase↗
PubMed1997

Ubiquitin C-terminal hydrolase is an immediate-early gene essential for long-term facilitation in Aplysia.

The switch from short-term to long-term facilitation of the synapses between sensory and motor neurons mediating gill and tail withdrawal reflexes in Aplysia requires CREB-mediated transcription and new protein synthesis. We isolated several downstream genes, one of which encodes a neuron-specific ubiquitin C-terminal hydrolase. This rapidly induced gene encodes an enzyme that associates with the proteasome and increases its proteolytic activity. This regulated proteolysis is essential for long-term facilitation. Inhibiting the expression or function of the hydrolase blocks induction of long-term but not short-term facilitation. We suggest that the enhanced proteasome activity increases degradation of substrates that normally inhibit long-term facilitation. Thus, through induction of the hydrolase and the resulting up-regulation of the ubiquitin pathway, learning recruits a regulated form of proteolysis that removes inhibitory constraints on long-term memory storage.

Amino Acid Sequence↗
PubMed1995

Cytomegalovirus-mediated induction of antisense mRNA expression to UL44 inhibits virus replication in an astrocytoma cell line: identification of an essential gene.

We have used an antisense RNA approach in the analysis of gene function in human cytomegalovirus (HCMV). An astrocytoma cell line (U373-MG) that is permissive for virus replication was permanently transfected with a construct bearing sequence from HCMV UL44 (coding for the major late DNA-binding protein, ppUL44, also known as pp52 or ICP36) in an antisense orientation and under the control of the immediate-early enhancer-promoter element. Upon HCMV infection at a high multiplicity, we found a marked reduction in UL44 protein products (the ICP36 family of proteins) in established cell transfectants and a strong inhibition of virus yield in infected-cell supernatants at two weeks postinfection, while herpes simplex virus replication was not affected. In infected cells, viral DNA replication was strongly inhibited. While gene products such as pUS22 and pUL32 were also inhibited, pUL123 and pUL82 accumulated in the infected cells over time. Our data suggest an essential role for the UL44 family of proteins in HCMV replication and represent a model of virus inhibition by virus-induced antisense RNA synthesis in genetically modified cells.

Animals↗
PubMed1992

MPI1, an essential gene encoding a mitochondrial membrane protein, is possibly involved in protein import into yeast mitochondria.

To identify components of the mitochondrial protein import pathway in yeast, we have adopted a positive selection procedure for isolating mutants disturbed in protein import. We have cloned and sequenced a gene, termed MPI1, that can rescue the genetic defect of one group of these mutants. MPI1 encodes a hydrophilic 48.8 kDa protein that is essential for cell viability. Mpi1p is a low abundance and constitutively expressed mitochondrial protein. Mpi1p is synthesized with a characteristic mitochondrial targeting sequence at its amino-terminus, which is most probably proteolytically removed during import. It is a membrane protein, oriented with its carboxy-terminus facing the intermembrane space. In cells depleted of Mpi1p activity, import of the precursor proteins that we tested thus far, is arrested. We speculate that the Mpi1 protein is a component of a proteinaceous import channel for translocation of precursor proteins across the mitochondrial inner membrane.

Amino Acid Sequence↗
PubMed1988

MAS1, a gene essential for yeast mitochondrial assembly, encodes a subunit of the mitochondrial processing protease.

We have previously described a yeast mutant (mas1) that accumulates mitochondrial precursor proteins at high temperature and is deficient in the activity of a matrix-localized protease which cleaves presequences from mitochondrial precursor proteins. We have now cloned and sequenced the wild-type MAS1 gene and found that it encodes a subunit of the mitochondrial processing protease, that it is essential for cell viability and that the protein product participates in its own cleavage during import into mitochondria. The MAS1 protein is thus the first genetically defined component of the mitochondrial protein import pathway.

Amino Acid Sequence↗
PubMed2005

Novel cellular genes essential for transformation of endothelial cells by Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is involved in the development of lymphoproliferative diseases and Kaposi's sarcoma. The oncogenicity of this virus is reflected in vitro by its ability to transform B cells and endothelial cells. Infection of dermal microvascular endothelial cells (DMVEC) transforms the cells from a cobblestone-like monolayer to foci-forming spindle cells. This transformation is accompanied by dramatic changes in the cellular transcriptome. Known oncogenes, such as c-Kit, are among the KSHV-induced host genes. We previously showed that c-Kit is an essential cellular component of the KSHV-mediated transformation of DMVEC. Here, we test the hypothesis that the transformation process can be used to discover novel oncogenes. When expression of a panel of KSHV-induced cellular transcripts was inhibited with antisense oligomers, we observed inhibition of DMVEC proliferation and foci formation using antisense molecules to RDC1 and Neuritin. We further showed that transformation of KSHV-infected DMVEC was inhibited by small interfering RNA directed at RDC1 or Neuritin. Ectopic expression of Neuritin in NIH 3T3 cells resulted in changes in cell morphology and anchorage-independent growth, whereas RDC1 ectopic expression significantly increased cell proliferation. In addition, both RDC1- and Neuritin-expressing cells formed tumors in nude mice. RDC1 is an orphan G protein-coupled receptor, whereas Neuritin is a growth-promoting protein known to mediate neurite outgrowth. Neither gene has been previously implicated in tumorigenesis. Our data suggest that KSHV-mediated transformation involves exploitation of the hitherto unrealized oncogenic properties of RDC1 and Neuritin.

Adaptor Proteins, Signal Transducing↗
PubMed1996

A new method for the identification and isolation of genes essential for Arabidopsis thaliana seed germination.

Seed viability, dormancy and germination efficiency are very important aspects of the life cycle of plants and their potential to survive and spread in the environment. To characterize the genes controlling these processes, we have devised a technique for the selection of mutants impaired in seed germination. Selection for such a trait is complicated by physiological factors that interact with these processes and affect seed germination efficiency. The distinction between low seed germination potential due to physiological factors that interfere with seed maturation or germination and germination deficiency due to genetic factors was based on screening for tagged mutations. Arabidopsis thaliana T-DNA primary transformants obtained by an in planta transformation technique are all heterozygotes. We screened for lack of germination of 1/4 of the seeds in the progeny of independent transformants, and simultaneously for the abnormal segregation (2:1 instead of 3:1) of a kanamycin resistance marker carried by the T-DNA inserted into the genome of these primary transformants in the plants that germinate. This yielded several mutants affected in the germination processes. One of the mutants, designated ABC33, was further characterized. Once the viable embryos from non-germinating seeds were removed from their testa, they grew and displayed a dwarf phenotype which could be complemented by providing gibberellic acid. A genetic and molecular analysis, based on the characterization of the flanking genomic sequences of the T-DNA insert, showed that ABC33 is a new loss-of-function allele at the GA1 locus.

Arabidopsis↗
PubMed1997

A gene essential for de novo methylation and development in Ascobolus reveals a novel type of eukaryotic DNA methyltransferase structure.

Molecular mechanisms determining methylation patterns in eukaryotic genomes still remain unresolved. We have characterized, in Ascobolus, a gene for de novo methylation. This novel eukaryotic gene, masc1, encodes a protein that has all motifs of the catalytic domain of eukaryotic C5-DNA-methyltransferases but is unique in that it lacks a regulatory N-terminal domain. The disruption of masc1 has no effect on viability or methylation maintenance but prevents the de novo methylation of DNA repeats, which takes place after fertilization, through the methylation induced premeiotically (MIP) process. Crosses between parents harboring the masc1 disruption are arrested at an early stage of sexual reproduction, indicating that the activity of Masc1, the product of the gene, is crucial in this developmental process.

Amino Acid Sequence↗
PubMed1988

Delimitation of essential genes of cassava latent virus DNA 2.

Insertion and deletion mutagenesis of both extended open reading frames (ORFs) of cassava latent virus DNA 2 destroys infectivity. Infectivity is restored by coinoculating constructs that contain single mutations within different ORFs. Although frequent intermolecular recombination produces dominant parental-type virus, mutants can be retained within the virus population indicating that they are competent for replication and suggesting that rescue can occur by complementation of trans acting gene products. By cloning specific fragments into DNA 1 coat protein deletion vectors we have delimited the DNA 2 coding regions and provide substantive evidence that both are essential for virus infection. Although a DNA 2 component is unique to whitefly-transmitted geminiviruses, the results demonstrate that neither coding region is involved solely in insect transmission. The requirement for a bipartite genome for whitefly-transmitted geminiviruses is discussed.

Amino Acid Sequence↗
PubMed1991

A nuclear factor NF-GM2 that interacts with a regulatory region of the GM-CSF gene essential for its induction in responses to T-cell activation: purification from human T-cell leukemia line Jurkat cells and similarity to NF-kappa B.

Activation of T cells by antigen, lectin, or a combination of phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) leads to the induction of genes for a set of lymphokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF). We demonstrated in earlier studies that the upstream region of the mouse GM-CSF promoter at positions between -95 and -73 is essential for transcriptional activation in response to PMA/A23187. This region contains two DNA-binding motifs, GM2 and GC-box. The GM2 sequence (GGTAGTTCCC) is recognized by an inducible factor NF-GM2; the other (CCGCCC) by constitutive factors A1, A2, and B. To elucidate the mechanism of GM-CSF gene activation, we have purified the inducible factor NF-GM2 from the nuclear extract of stimulated Jurkat cells on the basis of specific DNA-binding activity. The purified NF-GM2 consists of 50 (p50) and 65 kDa (p65) polypeptides and has a binding activity specific for both the GM-CSF and immunoglobulin kappa (GGAAAGTCCC) enhancers. Electrophoretically purified p50 alone can form a protein-DNA complex, but in the mixture, p50 associates preferentially with p65 to form the NF-GM2 complex. In addition, p65 gave per se, with low affinity, a protein-DNA complex that migrated more slowly than native NF-GM2 complex. Furthermore, an antiserum against KBF1 (identical to 50 kDa NF-kappa B protein) reacted with the p50 of NF-GM2, indicating that the NF-GM2 polypeptide cannot be immunologically differentiated from the 50 kDa subunit of NF-kappa B. The purified NF-GM2 activated in vitro transcription from the kappa B enhancer, while it failed to stimulate transcription from the GM-CSF promoter harboring the GM2 sequence. This suggests that the activation mechanism of the GM-CSF gene through the GM2/GC-box sequence is different from that of genes carrying the kappa B enhancer alone.

Base Sequence↗
PubMed1996

hiiragi, a gene essential for wing development in Drosophila melanogaster, affects the Notch cascade.

A recessive mutation, hiiragiP1, on the second chromosome of Drosophila was obtained by P element insertion mutagenesis. Flies homozygous for hiiragiP1 have notched wing margins. Genetic interactions between hiiragi and the genes that encode components of Notch signaling, such as Notch, Hairless, Serrate and deltex, strongly support the involvement of hiiragi in the signal transduction cascade of Notch. It has been reported that Serrate and Delta, other components of Notch signaling, share EGF-like repeats and a second conserved cysteine-rich motif, and that these components interact physically with the same region of Notch. In hiiragiP1; SerrateD/+ double mutants, we observed synergistic enhancement of the notched phenotype of wing margins. In contrast, Delta FX3 had no phenotypic effect on hiiragiP1 in hiiragiP1; Delta FX3/+ double mutants. Taken together, these results indicate that hiiragi is involved in the Notch signaling cascade induced by Serrate rather than by Delta.

Animals↗
PubMed1996

MHP1, an essential gene in Saccharomyces cerevisiae required for microtubule function.

The gene for a microtubule-associated protein (MAP), termed MHP1 (MAP-Homologous Protein 1), was isolated from Saccharomyces cerevisiae by expression cloning using antibodies specific for the Drosophila 205K MAP. MHP1 encodes an essential protein of 1,398 amino acids that contains near its COOH-terminal end a sequence homologous to the microtubule-binding domain of MAP2, MAP4, and tau. While total disruptions are lethal, NH2-terminal deletion mutations of MHP1 are viable, and the expression of the COOH-terminal two-thirds of the protein is sufficient for vegetative growth. Nonviable deletion-disruption mutations of MHP1 can be partially complemented by the expression of the Drosophila 205K MAP. Mhp1p binds to microtubules in vitro, and it is the COOH-terminal region containing the tau-homologous motif that mediates microtubule binding. Antibodies directed against a COOH-terminal peptide of Mhp1p decorate cytoplasmic microtubules and mitotic spindles as revealed by immunofluorescence microscopy. The overexpression of an NH2-terminal deletion mutation of MHP1 results in an accumulation of large-budded cells with short spindles and disturbed nuclear migration. In asynchronously growing cells that overexpress MHP1 from a multicopy plasmid, the length and number of cytoplasmic microtubules is increased and the proportion of mitotic cells is decreased, while haploid cells in which the expression of MHP1 has been silenced exhibit few microtubules. These results suggest that MHP1 is essential for the formation and/or stabilization of microtubules.

Amino Acid Sequence↗
PubMed1994

The Haemophilus influenzae sxy-1 mutation is in a newly identified gene essential for competence.

The sxy-1 mutation of Haemophilus influenzae causes a 100- to 1,000-fold increase in spontaneous natural competence. We have used mapping and sequencing to identify this mutation as a G-to-A transition in an open reading frame adjacent to the rec-1 locus. This mutation substitutes valine for isoleucine at amino acid 19 of the protein specified by this gene (now named sxy). A multicopy plasmid containing the wild-type sxy gene confers constitutive competence on wild-type cells. Cells carrying this plasmid exhibit, in all stages of growth, DNA uptake levels and transformation frequencies as high those normally seen only after full induction of competence by starvation; deletion of part of the sxy gene from the plasmid abolishes this effect. In contrast, a transposon insertion in sxy entirely prevents both DNA uptake and transformation, indicating that sxy encodes a function essential for competence. These findings suggest that sxy may act as a positive regulator of competence. However, because cells carrying the transposon-inactivated sxy::Tn allele grow slowly under conditions that do not induce competence, sxy may also have a role in noncompetent cells.

Alleles↗
PubMed2002

Transcriptional regulation of the membrane-associated prostaglandin E2 synthase gene. Essential role of the transcription factor Egr-1.

Membrane-associated prostaglandin (PG) E2 synthase (mPGES) is an inducible terminal enzyme in the biosynthetic pathway for prostaglandin E2, which participates in many biological processes. In this study, we investigated the molecular mechanism controlling the inducible expression of mPGES. The mouse mPGES gene consisted of three exons, and its 5'-proximal promoter contained consensus motifs for the binding of several transcription factors. Transgenic expression in mice of the mouse mPGES promoter flanked by a reporter gene resulted in stimulus-dependent induction of the reporter in tissues where mPGES was intrinsically induced. Deletion and site-specific mutation analyses of the 5'-flanking region demonstrated that stimulus-inducible expression of mouse and human mPGES required tandem GC boxes adjacent to the initiation site. The stimulus-induced GC box binding activity was present in nuclear extracts of cells, in which the proximal GC box was essential for binding. An 80-kDa stimulus-inducible nuclear protein that bound to this GC box was identified as the transcription factor Egr-1 (for early growth response-1). These results suggest that Egr-1 is a key transcription factor in regulating the inducible expression of mPGES.

Amino Acid Motifs↗