Functional characterization of the zebrafish WHSC1-related gene, a homolog of human NSD2.

Methylation of specific lysine residues of histone H3 and H4 has been reported to be important in the structuring of chromatin and for the transcription of certain genes. Proteins with SET domains have been shown to methylate specific lysine residues of histone H3 and H4. We isolated a SET domain-containing gene from the zebrafish (Danio rerio).

Decreased serum dependence in the growth of NIH3T3 cells from the overexpression of human nuclear receptor-binding SET-domain-containing protein 1 (NSD1) or fission yeast su(var)3-9, enhancer-of-zeste, trithorax 2 (SET2).

Nuclear receptor-binding SET-domain-containing protein 1 (NSD1), a culprit gene for Sotos syndrome, contains a su(var)3-9, enhancer-of-zeste, trithorax (SET) domain that is responsible for histone methyltransferase activity and other domains such as plant homeodomain (PHD) and proline-tryptophan-tryptophan-proline (PWWP) involved in protein-protein interactions in the C-terminal half of NSD1. To elucidate the function of NSD1 on cell growth, we overexpressed NSD1 in NIH3T3 cells. Cells overexpressing NSD1 grew in the presence of 2% serum, whereas vector transfected cells did not.

Crystal structure of uridine-diphospho-N-acetylglucosamine pyrophosphorylase from Candida albicans and catalytic reaction mechanism.

Uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc) is a precursor of the bacterial and fungal cell wall. It is also used in a component of N-linked glycosylation and the glycosylphosphoinositol anchor of eukaryotic proteins. It is synthesized from N-acetylglucosamine-1-phosphate (GlcNAc-1-P) and uridine-5'-triphosphate (UTP) by UDP-GlcNAc pyrophosphorylase (UAP).

Purification, crystallization and preliminary X-ray diffraction studies of UDP-N-acetylglucosamine pyrophosphorylase from Candida albicans.

UDP-N-acetylglucosamine pyrophosphorylase (UAP) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine. UAP from Candida albicans was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals of the substrate and product complexes both diffract X-rays to beyond 2.

Crystal structures of N-acetylglucosamine-phosphate mutase, a member of the alpha-D-phosphohexomutase superfamily, and its substrate and product complexes.

N-acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthetic process of UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a UDP sugar that serves as a biosynthetic precursor of glycoproteins, mucopolysaccharides, and the cell wall of bacteria. Thus, a specific inhibitor of AGM1 from pathogenetic fungi could be a new candidate for an antifungal reagent that inhibits cell wall synthesis.

Purification, crystallization and preliminary X-ray diffraction studies of N-acetylglucosamine-phosphate mutase from Candida albicans.

N-acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc) in eukaryotes and belongs to the alpha-D-phosphohexomutase superfamily. AGM1 from Candida albicans (CaAGM1) was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals obtained belong to the primitive monoclinic space group P2(1), with unit-cell parameters a = 60.

Modulation at a cellular level of the thyroid hormone receptor-mediated gene expression by 1,2,5,6,9,10-hexabromocyclododecane (HBCD), 4,4'-diiodobiphenyl (DIB), and nitrofen (NIP).

Previously, we demonstrated that some endocrine disrupting chemicals affected thyroid hormone receptor (TR)-mediated gene expression in HeLaTR cells that stably expressed the human TRalpha1. To examine whether widely used brominated flame retardants and pesticides affect TR-mediated gene expression, those with organohalogen, which is also present in T3, were screened. To monitor the TR-mediated gene expression, HeLaTR cells were transfected with a luciferase gene that was linked to the thyroid hormone responsive element.

2,3,7,8-tetrachlorodibenzo-p-dioxin augments the modulation of gene expression mediated by the thyroid hormone receptor.

Previously, we reported on genes whose expression was highly modulated by T3 in the HeLaTR cells that stably expressed the thyroid hormone receptor (TR). In this study, we examined the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on TR-mediated gene expression. In the HeLaTR cells, T3 induced the expression of the reporter gene in a thyroid hormone responsible element (TRE)-dependent manner.

Thyroid hormone induces the expression of 4-1BB and activation of caspases in a thyroid hormone receptor-dependent manner.

Thyroid hormone has various effects on cell proliferation, growth and apoptosis. To gain more insight into the molecular dynamics caused by thyroid hormone, gene expression in HeLaTR cells that constitutively overexpressed the thyroid hormone receptor (TR) was analyzed. Gene expression profiling of the HeLaTR cells with an oligonucleotide microarray yielded 229 genes whose expression was significantly altered by T3.

Isolation and functional analysis of a gene, tcsB, encoding a transmembrane hybrid-type histidine kinase from Aspergillus nidulans.

We cloned and characterized a novel Aspergillus nidulans histidine kinase gene, tcsB, encoding a membrane-type two-component signaling protein homologous to the yeast osmosensor synthetic lethal N-end rule protein 1 (SLN1), which transmits signals through the high-osmolarity glycerol response 1 (HOG1) mitogen-activated protein kinase (MAPK) cascade in yeast cells in response to environmental osmotic stimuli. From an A. nidulans cDNA library, we isolated a positive clone containing a 3,210-bp open reading frame that encoded a putative protein consisting of 1,070 amino acids.

Characterization of the CaNAG3, CaNAG4, and CaNAG6 genes of the pathogenic fungus Candida albicans: possible involvement of these genes in the susceptibilities of cytotoxic agents.

CaNAG3, CaNAG4, and CaNAG6 form a gene cluster with CaNAG1 CaNAG2 and CaNAG5 that are responsible for glucosamine-6-phosphate deaminase, N-acetylglucosamine-phosphate deacetylase and N-acetylglucosamine kinase, but their functions largely remain unclear. In this study, Candida albicans cells carrying null mutations in either CaNAG3, CaNAG4, or CaNAG6 were generated and characterized. They showed increased susceptibility to cycloheximide and attenuated virulence in mice.

Identification and characterization of the genes for N-acetylglucosamine kinase and N-acetylglucosamine-phosphate deacetylase in the pathogenic fungus Candida albicans.

Like bacteria and many fungi, the pathogenic fungus Candida albicans can utilize GlcNAc as a carbon source for growth. A cluster of six genes was identified in the C. albicans genome.

Functional cloning and mutational analysis of the human cDNA for phosphoacetylglucosamine mutase: identification of the amino acid residues essential for the catalysis.

In Saccharomyces cerevisiae, phosphoacetylglucosamine mutase is encoded by an essential gene called AGM1. The human AGM1 cDNA (HsAGM1) and the Candida albicans AGM1 gene (CaAGM1) were functionally cloned and characterized by using an S. cerevisiae strain in which the endogenous phosphoacetylglucosamine mutase was depleted.

The yeast Chs4 protein stimulates the trypsin-sensitive activity of chitin synthase 3 through an apparent protein-protein interaction.

Inducible overexpression of the CHS4 gene under the control of the GAL1 promoter increased Chs3p (chitin synthase 3) activity in Saccharomyces cerevisiae several fold. Approximately half of the Chs3p activity in the membranes of cells overexpressing Chs4p was extracted using CHAPS and cholesteryl hemisuccinate. The detergent-extractable Chs3p activity appeared to be non-zymogenic because incubation with trypsin decreased enzyme activity in both the presence and absence of the substrate, UDP-N-acetylglucosamine.

The Candida albicans gene for mRNA 5-cap methyltransferase: identification of additional residues essential for catalysis.

The 5'-cap structure of eukaryotic mRNA is methylated at the terminal guanosine by RNA (guanine-N7-)-methyltransferase (cap MTase). Saccharomyces cerevisiae ABD1 (ScABD1) and human hMet (also called CMT1) genes are responsible for this enzyme. The ABD1 homologue was cloned from the pathogenic fungus Candida albicans and named C.

Roles of three histidine kinase genes in hyphal development and virulence of the pathogenic fungus Candida albicans.

The pathogenic fungus Candida albicans harbors three histidine kinase genes called CaSLN1, CaNIK1, and CaHK1. The disruption of any one of these three genes impaired the hyphal formation and attenuated the virulence of C. albicans in a mouse systemic candidiasis model.

Mutational analysis of chitin synthase 2 of Saccharomyces cerevisiae. Identification of additional amino acid residues involved in its catalytic activity.

Saccharomyces cerevisiae harbors three chitin synthases termed Chs1p, Chs2p and Chs3p. Previously, we demonstrated that con1, a region that is highly conserved among all chitin synthases, contains amino acids essential for the catalytic activity of the enzyme and that Asp562, Gln601, Arg604, and Trp605 found in con1 together with Asp441 were probable catalytic sites of the enzyme. Here we report that another region, con2, in the C-terminal half of Chs2p is also conserved exclusively in chitin synthases that resemble S.

Saccharomyces cerevisiae GNA1, an essential gene encoding a novel acetyltransferase involved in UDP-N-acetylglucosamine synthesis.

The Saccharomyces cerevisiae gene, YFL017C, for a putative acetyltransferase was characterized. Disruption of YFL017C was lethal, leading to a morphology similar to those caused by the depletion of AGM1 or UAP1, the genes encoding phospho-N-acetylglucosamine mutase and UDP-N-acetylglucosamine pyrophosphorylase, respectively. This implies the involvement of YFL017C in UDP-N-acetylglucosamine synthesis.

Isolation and characterization of the Candida albicans gene for mRNA 5'-triphosphatase: association of mRNA 5'-triphosphatase and mRNA 5'-guanylyltransferase activities is essential for the function of mRNA 5'-capping enzyme in vivo.

The amino acid sequence of the Saccharomyces cerevisiae mRNA 5'-triphosphatase (TPase) diverges from those of higher eukaryotes. In order to confirm the sequence divergence of TPases in lower and higher eukaryotes, the Candida albicans gene for TPase was identified and characterized. This gene designated CaCET1 (C.

Isolation and characterization of a human cDNA for mRNA 5'-capping enzyme.

The human mRNA 5'-capping enzyme cDNA was identified. Three highly related cDNAs, HCE1 (human mRNAcappingenzyme1), HCE1A and HCE1B , were isolated from a HeLa cDNA library. The HCE1 cDNA has the longest ORF, which can encode a 69 kDa protein.

Isolation of CaSLN1 and CaNIK1, the genes for osmosensing histidine kinase homologues, from the pathogenic fungus Candida albicans.

Recent studies have revealed that fungi possess a mechanism similar to bacterial two-component systems to respond to extracellular changes in osmolarity. In Saccharomyces cerevisiae, Sln1p contains both histidine kinase and receiver (response regulator) domains and acts as an osmosensor protein that regulates the downstream HOG1 MAP kinase cascade. SLN1 of Candida albicans was functionally cloned using an S.

Cloning of the Candida albicans homolog of Saccharomyces cerevisiae GSC1/FKS1 and its involvement in beta-1,3-glucan synthesis.

Saccharomyces cerevisiae GSC1 (also called FKS1) and GSC2 (also called FKS2) have been identified as the genes for putative catalytic subunits of beta-1,3-glucan synthase. We have cloned three Candida albicans genes, GSC1, GSL1, and GSL2, that have significant sequence homologies with S. cerevisiae GSC1/FKS1, GSC2/FKS2, and the recently identified FKSA of Aspergillus nidulans at both nucleotide and amino acid levels.

Isolation of the Candida albicans homologs of Saccharomyces cerevisiae KRE6 and SKN1: expression and physiological function.

Cell wall beta-glucan in a pathogenic fungus, Candida albicans, is highly branched with beta-1,3 and beta-1,6 linkages. We have isolated the C. albicans cDNAs for KRE6 and SKN1, the genes required for beta-1,6-glucan synthesis in Saccharomyces cerevisiae.

Isolation of the mRNA-capping enzyme and ferric-reductase-related genes from Candida albicans.

The mRNA-capping enzyme (mRNA 5'-guanylyltransferase) gene was cloned from a Candida albicans genomic DNA library by functional complementation of a Saccharomyces cerevisiae ceg1 delta null mutation. This gene, referred to as CGT1 (C. albicans guanylyltransferase 1), can encode a 52 kDa protein that is highly homologous to S.

Normal and transforming Ras are differently regulated for posttranslational modifications.

Point mutation of the c-H-ras gene significantly increases cellular transforming activities of Ras. Since posttranslational modification and subsequent membrane localization are essential for the biological activities of Ras, we examined whether or not the mutation also affects these two factors. The normal (Gly(12)) or the transforming (Val(12)) c-H-ras gene was expressed in NIH3T3 cells using a metallothionein promoter.