1.1.1.6 glycerol dehydrogenase molecular biology enzymatic redox cofactor regeneration in organic media: functionalization and application of recombinant glycerol dehydrogenase and soluble transhydrogenase in reverse micelles, overview 1.1.1.9 D-xylulose reductase molecular biology a high thermostability of PsXDH is obtained by subsequent site-directed mutagenesis of the structural zinc-binding loop. The best mutant in this study (C4/F98R/E101F) shows a 10.8 °C higher thermal transition temperature and 20.8 °C higher half denaturation temperature (T1/2) compared with wild-type 1.1.1.22 UDP-glucose 6-dehydrogenase molecular biology the high activity combined with the simple purification procedure used make GbUGD a valuable alternative biocatalyst for the synthesis of UDP-glucuronic acid or the development of NAD+ regeneration systems 1.1.1.28 D-lactate dehydrogenase molecular biology D-lactate dehydrogenase is useful as a marker gene allowing positive selection of transgenic plants 1.1.1.34 hydroxymethylglutaryl-CoA reductase (NADPH) molecular biology Lactococcus lactis is a potential heterologous host for the production of sesquiterpenes from a herbaceous Malaysian plant, Persicaria minor. A sesquiterpene synthase gene encoding beta-sesquiphellandrene synthase from Persicaria minor is successfully cloned and expressed in Lactococcus lactis. Overexpression of the Lactococcus lactis endogenous 3-hydroxy-3-methylglutaryl Co-A reductase, an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increases the production level of beta-sesquiphellandrene by 1.25-1.60 fold 1.1.1.95 phosphoglycerate dehydrogenase molecular biology results demonstrate that PGDH enhances the levels of betaine by providing the precursor serine for both choline oxidation and glycine methylation pathways 1.1.1.95 phosphoglycerate dehydrogenase molecular biology results demonstrate that the promoter activity of the human PHGDH gene is positively regulated by the action of transcription factors Sp1 and NF-Y 1.1.1.188 prostaglandin-F synthase molecular biology genome editing of human cell lines can be used to complement mouse KO models to validate the function of genes in differentiated tissues and cells 1.1.1.219 dihydroflavonol 4-reductase molecular biology the gene encoding dihydroflavonol 4-reductase is a candidate for the anthocyaninless locus of rapid cycling Brassica rapa (fast plants type) 1.1.3.8 L-gulonolactone oxidase molecular biology short-term vitamin A deficiency in broiler chicks reduces GULO activity without concomittant changes in tissue ascorbic acid 1.1.3.9 galactose oxidase molecular biology glycoprotein labeling using engineered variants of galactose oxidase, overview 1.1.3.13 alcohol oxidase molecular biology Pichia pastoris is an efficient host for the expression and secretion of heterologous proteins possessing a strong and tightly regulated promoter from the alcohol oxidase I, AOX1, gene. The transformed cells need to be activated by methanol and grow on methanol as carbon source. With the inducible AOX1 promoter an increase of the copy number above two resulted in a decrease of expression. Combined use of GAP and AOX1 promoters in Pichia pastoris, overview 1.1.3.15 (S)-2-hydroxy-acid oxidase molecular biology the interaction of capsid protein P8 with the GOX of host cells leads to translocation of capsid protein P8 into peroxisomes. The interaction between capsid protein P8 and GOX plays important roles in Rice dwarf phytoreovirus targeting into the replication site of host cells 1.1.98.7 Ser-type anaerobic sulfatase-maturating enzyme molecular biology enzymatic modification of a DARPin model protein carrying the aldehyde tag sequence SAPAR enables the production of a fluorescent conjugate. Thus proving its potential for bioconjugation chemistry. This tag system could be useful in orthogonal combination with the standard cysteine-type tag system targeted by formylglycine-generating enzyme, which is regularly utilized for protein labeling 1.1.99.29 pyranose dehydrogenase (acceptor) molecular biology gene expression analysis using PCR, pdh1 expression is upregulated upon exhaustion of the carbon source and appears to be additionally regulated under conditions of oxygen limitation, PDH production is highest on cellobiose and very low on fructose 1.1.99.29 pyranose dehydrogenase (acceptor) molecular biology gene expression analysis using PCR, pdh2 is constitutively expressed, PDH production is highest on cellobiose and very low on fructose 1.1.99.29 pyranose dehydrogenase (acceptor) molecular biology gene expression analysis using PCR, pdh3 is constitutively expressed, PDH production is highest on cellobiose and very low on fructose 1.2.1.8 betaine-aldehyde dehydrogenase molecular biology BADH application as a marker for chloroplast engineering without using antibiotic can avoid transferring antibiotic genes from the plant and thus assists to allay public concern regarding genetic modifications 1.2.1.36 retinal dehydrogenase molecular biology ALDH1A1 is rapidly gaining importance as a stem cell marker 1.3.1.77 anthocyanidin reductase [(2R,3R)-flavan-3-ol-forming] molecular biology a method for the analysis of ANR activity using the detection of coenzyme is established 1.3.5.1 succinate dehydrogenase molecular biology considering the conservation of amino acids crucial for the SQR activity and the high levels of ROS production from the mitochondrial complex II of the Ascaris suum adult worm together with the absence of complexes III and IV activities in its respiratory chain, it is a good model to examine the reactive oxygen species production from the mitochondrial complex II 1.3.5.1 succinate dehydrogenase molecular biology the iron-sulfur subunit (SdhB) of mitochondrial succinate dehydrogenase is encoded by a split and rearranged nuclear gene in Euglena gracilis. The two subgenic modules are transcribed independently and the resulting mRNAs appear to be independently translated, with the two protein products imported into mitochondria. The discovery of this unique molecular marker provides evidence for the monophyly of Euglenozoa that is independent of evolutionary models 1.3.5.1 succinate dehydrogenase molecular biology the relevance of modifications in Alternaria alternata AaSdhB sequence in conferring boscalid resistance is discussed 1.3.5.1 succinate dehydrogenase molecular biology the splitting of sdhB in Euglena and trypanosomatids is an example of a unique molecular character that specifically unites these two phylogenetic groups 1.3.99.4 3-oxosteroid 1-dehydrogenase molecular biology development of a synthetic 3-ketosteroid DELTA1-dehydrogenase for the generation of a catabolic pathway enabling cholesterol degradation in human cells 1.4.1.2 glutamate dehydrogenase molecular biology GDH is essential for the full development of the secretory response in beta-cells 1.4.1.2 glutamate dehydrogenase molecular biology GDH, in conjunction with NADH-glutamte synthase, contributes to the control of leaf glutamate homeostasis, an amino acid that plays a central signaling and metabolic role at the interface of the carbon and nitrogen assimilatory pathways 1.4.1.4 glutamate dehydrogenase (NADP+) molecular biology promoter of GDH from Xanthophyllomyces dendrorhous is shown to be a valuable tool for controlled gene expression in Basidiomycetes 1.4.1.4 glutamate dehydrogenase (NADP+) molecular biology results implicate glutamate dehydrogenase and NADP-GDH in particular, as a key target of in vivo isophthalate inhibition during ammonium assimilation 1.4.1.13 glutamate synthase (NADPH) molecular biology GltA shows 73% identity to the corresponding protein of Oceanobacillus iheyensis 1.4.1.13 glutamate synthase (NADPH) molecular biology GltB1 shows 74% identity to the corresponding protein of Oceanobacillus iheyensis 1.4.1.13 glutamate synthase (NADPH) molecular biology GltB2 shows 73% identity to the glutamate synthase of Geobacillus kaustophilus 1.4.1.13 glutamate synthase (NADPH) molecular biology sequence identity between Salmonella typhimurium and Escherichia coli GOGAT is 91%, Salmonella typhimurium GOGAT region can complement an Escherichia coli mutant defective in GOGAT 1.4.3.1 D-aspartate oxidase molecular biology This enzyme is proposed to have a role in the inactivation of the synaptically released D-aspartate. Its C-terminus has a peroxisome targeting signal. 1.5.1.11 D-octopine dehydrogenase molecular biology His-tag-induced crystallization of OcDH is found to be dependent on the length of the His tag 1.8.1.15 mycothione reductase molecular biology a DTNB-coupled assay is developed for time-dependent inhibition of Mycobacterium tubercolosis reductase employing a benzyl glycoside analogue of MSH, from which an efficient mixed disulfide substrate is chemically recycled in situ, thereby greatly reducing the substrate quantities needed for such assays 1.8.3.1 sulfite oxidase molecular biology molybdenum trioxide (MoO3) nanoparticles display an intrinsic biomimetic sulfite oxidase activity under physiological conditions, and, functionalized with a customized bifunctional ligand containing dopamine as anchor group and triphenylphosphonium ion as targeting agent, they selectively target the mitochondria while being highly dispersible in aqueous solutions. Chemically induced sulfite oxidase knockdown cells treated with MoO3 nanoparticles recover their sulfite oxidase activity in vitro, which makes MoO3 nanoparticles a potential therapeutic for sulfite oxidase deficiency and opens new avenues for cost-effective therapies for gene-induced deficiencies. Molybdenum trioxide (MoO3) is a well-known model compound for selective oxidation catalysis 1.13.11.49 chlorite O2-lyase molecular biology use of the heme enzyme for the rapid, in situ generation of O2 at concentrations far exceeding 2 mM in coupled enzyme reaction systems to study O consumption or O2 involving reaction steps. Catalytic concentrations of chlorite O2-lyase can be used to initiate the reaction of an O2-utilizing (metallo)enzyme by rapid mixing with the highly soluble, non-volatile ClO2, rather than with the sparingly soluble, gaseous O2, e.g. activation of the beta2 subunit of class Ic ribonucleotide reductase from Chlamydia trachomatis by mixing its MnII/FeII complex with ClO2- in the presence of chlorite O2-lyase, overview 1.13.12.5 Renilla-type luciferase molecular biology development of a reverse genetic model to characterize the pathway of replication and pathogenesis of the SARS coronavirus. Renilla luciferase is used as a reporter gene and inserted into the backbone of the infectious clone of SARS coronavirus to replace ORF 7a/b (SARS wt-Luc), which is believed to have apoptotic effects on host cells. Recombinant viruses with luciferase constructs are isolated and shown to stably maintain the Renilla luciferase gene and to express subgenomic mRNA encoding luciferase. SARS wt-Luc is a viable virus that allows studies of the effect of subgenomic manipulation on virus efficiacy, both in replication and subgenomic production. This approach offers an alternative to plaque assay analysis in testing the efficiency of anti-SARS agents 1.13.12.5 Renilla-type luciferase molecular biology dual luciferase enzyme assay system for reporter gene analysis combining both the firefly luciferase enzyme and the Renilla luciferase enzyme in a nonproprietary buffer 1.13.12.5 Renilla-type luciferase molecular biology modification of the photoprotein aequorin by attaching selected fluorophores at a unique site on the protein. This will allow for in vitro transfer of bioluminescent energy from aequorin to the fluorophore thus creating an artificial jellyfish. The fluorophores are selected such that the excitation spectrum of the fluorophore overlaps with the emission spectrum of aequorin. By modifying aequorin with different fluorophores, bioluminescent labels with different emission maxima are produced, which will allow for the simultaneous detection of multiple analytes 1.13.12.5 Renilla-type luciferase molecular biology Renilla luciferase, fused to biospecific sequences such as engineered antibodies, can be administered systemically to provide a novel, sensitive method for optical imaging based on expression of cell surface receptors in living organism 1.13.12.5 Renilla-type luciferase molecular biology the development of variants of Renilla luciferase, which exhibit significantly improved properties compared with the native enzyme, will allow enhanced sensitivity in existing luciferase-based assays as well as enable the development of novel probes labeled with the luciferase protein 1.13.12.5 Renilla-type luciferase molecular biology when aequorin is microinjected into cleavage-stage zebrafish embryos, it is largely used up by about 24 hours. Thus, it is not possible to image Ca2+ signals from later stages of zebrafish development using this approach. Transient expression of apoaequorin (i.e., the protein component of aequorin) using aeq-mRNA in zebrafish embryos and then reconstitution of intact aequorin in vivo by loading the coelenterazine cofactor into the embryos separately provides a valuable tool for monitoring Ca2+ signaling during the 24–48 h post fertilization period of zebrafish development. Thus, it effectively extends the aequorin-based Ca2+-imaging window by an additional 24 hours 1.13.12.5 Renilla-type luciferase molecular biology sensitive reproter for studies of gene expression, promoter activity, protein-protein interactions, signal transduction, tumor cell growth, response to therapy 1.13.12.5 Renilla-type luciferase molecular biology an advanced Fc-binding probe, FcUni-RLuc, is produced and functionally assayed for labelling IgGs. The Fc antibody binding sequence HWRGWV is fused to Renilla luciferase, and the purified probe is employed for bioluminescence enzyme-linked immunoabsorbance assay of Her2 positive cells 1.13.12.5 Renilla-type luciferase molecular biology Renilla luciferase (Rluc) from Renilla reniformis is an appropriate protein reporter for the detection of specific molecular targets due to its bioluminescent feature, although its relatively low stability limits the application 1.13.12.6 Cypridina-luciferin 2-monooxygenase molecular biology construction of a cold-induced expression vector (pCold-ZZ-VL vector) in Escherichia coli cells that results in soluble bioluminescent fusion enzyme with binding ability to monoclonal antibodies, however, the Cypridina luciferase fusion enzyme is soluble but not bioluminescent (in contrast to other luciferases fused to the ZZ-domain of Staphylococcuss aureus protein A) 1.13.12.6 Cypridina-luciferin 2-monooxygenase molecular biology Renilla and Cypridina luciferases should be more appropriate tools for applications requiring the detection of small amounts of substrate as in small molecule detection assays because RLuc and CLuc respond to their luciferin concentration in a linear non-cooperative manner 1.13.12.6 Cypridina-luciferin 2-monooxygenase molecular biology Cypridina noctiluca luciferase is utilized for biochemical and molecular biological applications, including bioluminescent enzyme immunoassays, far-red luminescence imaging, and high-throughput reporter assays 1.13.12.6 Cypridina-luciferin 2-monooxygenase molecular biology luciferases can be used as light-emissive reporters of mechanoenzymatic reaction. The light emitted by a bioluminescent reaction can be used to directly monitor the progress of a mechanoenzymatic reaction without sampling 1.13.12.6 Cypridina-luciferin 2-monooxygenase molecular biology the thermostable enzyme can be used for various research applications, including in vivo imaging and high throughput reporter assays 1.13.12.7 firefly luciferase molecular biology Cypridina noctiluca luciferase is utilized for biochemical and molecular biological applications, including bioluminescent enzyme immunoassays, far-red luminescence imaging, and high-throughput reporter assays 1.13.12.7 firefly luciferase molecular biology firefly luciferase is widely used in molecular biology and bioanalytical systems as a reporter molecule due to the high quantum yield of the bioluminescence, availability of stable mutant forms of the enzyme with prescribed spectral characteristics and abundance of bacterial expression systems suitable for production of recombinant proteins in limitless quantities. Fusion proteins of luciferase are described with biotin-binding domain and treptavidin, with proteins A and G, antibodies, with DNA- and RNA-binding proteins, as well as fusion proteins designed for BRET systems. The firefly luciferase-based fusion proteins are represented as an effective tool for the development of different bioanalytical systems such as (1) systems in which luciferase is attached to the surface of the target and the bioluminescence signal is detected from the specific complexes formed, (2) BRET-based systems, in which the specific interaction induces changes in the bioluminescence spectrum, and (3) systems that use modified or split luciferases, in which the luciferase activity changes under the action of the analyte. All these systems have wide application in biochemical analysis of physiologically important compounds, for the detection of pathogenic bacteria and viruses, for evaluation of protein-protein interactions, assaying of metabolites involved in cell communication and cell signaling 1.13.12.7 firefly luciferase molecular biology Ppy luciferase can been used extensively as a reporter gene in living cells and organisms. Some biological applications are limited by the low stability of the luciferase and limited intracellular luciferin concentration. The mutant enzyme T214A/A215L/I232A/F295L/E345K/I423L/D436G/L530R exhibits both improved thermostability and brighter luminescence at low luciferin concentrations, it may be useful for reporter gene applications 1.13.12.7 firefly luciferase molecular biology the enzyme is a powerful tool for molecular and cellular biology, and popular in high-throughput screening and drug discovery 1.13.12.13 Oplophorus-luciferin 2-monooxygenase molecular biology useful reporter protein in various assay systems including reporter assays and immunoassays 1.13.12.13 Oplophorus-luciferin 2-monooxygenase molecular biology a multicolor BRET assay for the quantification of global DNA methylation is developed using CXXC-fused Oplophorus luciferase (CXXC-Oluc), a methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc), BOBO-1, and BOBO-3. CXXC-Oluc recognized unmethylated CpG sites on genomic DNA to excite BOBO-1 DNA intercalating dye, whereas MBD-Fluc recognized methylated CpG sites on genomic DNA to excite BOBO-3 DNA intercalating dye. The emission intensities of BOBO-1 and BOBO-3 are simultaneously detected and depended on the unmethylated and methylated CpG contents of the genomic DNA. There is a significant negative correlation between the emission intensities of BOBO-1 and BOBO-3. Therefore, the global DNA methylation level can be quantified with this multicolor BRET assay using a single tube 1.13.12.13 Oplophorus-luciferin 2-monooxygenase molecular biology bioluminescence imaging is a powerful, broadly utilized method for non-invasive imaging studies in cell-based assays and small animal models of normal physiology and multiple diseases. In combination with molecular engineering of cells and entire organisms using luciferase enzymes, bioluminescence imaging has enabled novel applications including studies of protein-protein interactions, ligand-receptor interactions, cell trafficking, and drug targeting in mouse models. The use of a luciferase enzyme derived from Oplophorus gracilirostris, NanoLuc, is described in cell-based assays bioluminescence imaging of tumor-bearing mice. NanoLuc is combined with another luciferase enzyme, firefly luciferase, to image multiple signal transduction events in one imaging session 1.14.11.1 gamma-butyrobetaine dioxygenase molecular biology a fluorescence assay based on the detection of fluoride released from reactions of fluorinated substrates with BBOX by the use of tert-butyldimethylsilyl-protected fluorescein is developed. (3S)-3-fluoro-4-(trimethylammonio)butanoate is a good substrate for BBOX, releasing fluoride when subjected to the enzyme 1.14.13.9 kynurenine 3-monooxygenase molecular biology development of a transformant selection system for Tribolium castaneum on the basis of mutant rescue 1.14.13.9 kynurenine 3-monooxygenase molecular biology wild-type KMO gene can be used as a marker gene for visually screening transgenic silkworms 1.14.13.9 kynurenine 3-monooxygenase molecular biology the wild-type enzyme gene can be used as a marker gene for visually screening transgenic silkworms 1.14.14.3 bacterial luciferase molecular biology enzyme can be used to monitor changes in gene expression as a reporter system in slow-growing mycobacteria, i.e. Mycobacterium tuberculosis strain H37Ra, determination of recombinant enzyme decay rate 1.14.14.3 bacterial luciferase molecular biology the enzyme is used as a reporter system tool for analysis of promoter and gene expression activity, overview 1.14.14.86 ent-kaurene monooxygenase molecular biology the fungal cytochrome P450 monooxygenase isolated from from Phaeoshaeria sp., strain L487 in the Pichia pastoris expression system might become a tool for functional assays of a variety of rice cytochrome P450 monooxygenases involved in the biosynthesis of secondary metabolites 1.14.16.1 phenylalanine 4-monooxygenase molecular biology an automated fluorescence-based continuous real-time PAH activity assay that is faster and more efficient but as precise and accurate as standard methods is developed 1.14.16.1 phenylalanine 4-monooxygenase molecular biology bicistronic expression system is developed which allows the isolation of hybrid forms that exhibit negative interallelic complementation, and may represent a model system for studying the molecular pathogenic mechanisms of PAH gene mutations in compound heterozygous phenylketonuric patients, providing the rationale to understand the observed inconsistencies both in genotype/phenotype correlations and in the response to (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin supplementation 1.17.1.4 xanthine dehydrogenase molecular biology isozyme XDH1 has the potential to be used as a metabolic target for controlling populations of Aedes aegypti mosquitoes, which are vectors of public health threats 1.17.4.1 ribonucleoside-diphosphate reductase molecular biology titration of ribonucleotide reductase expression can serve as a standard model system for studying the coordination between chromosome replication, cell division, and cell size 2.1.1.5 betaine-homocysteine S-methyltransferase molecular biology osmotic regulation of BHMT may be part of a cell volume-regulatory response and additionally lead to metabolic alterations that depend on the availability of betaine-derived methyl groups 2.1.1.5 betaine-homocysteine S-methyltransferase molecular biology S-adenosylmethionine and 5’-methylthioadenosine down-regulate BHMT expression in HepG2 cells in part by inducing NF-kappaB, which acts as a repressor for the human BHMT gene. While S-adenosylmethionine’s mechanism is NF-kappaB-dependent, 5’-methylthioadenosine has both NF-kappaB-dependent and -independent mechanisms 2.1.1.5 betaine-homocysteine S-methyltransferase molecular biology the BHMT/betaine system directly protects hepatocytes from homocysteine-induced injury but not tunicamycin-induced injury, including an endoplasmic reticulum stress response, lipid accumulation, and cell death 2.1.1.35 tRNA (uracil54-C5)-methyltransferase molecular biology enzyme is used for detecting tRNA-like moieties in viral RNA 2.1.1.63 methylated-DNA-[protein]-cysteine S-methyltransferase molecular biology modest binding cooperativity and high binding densities of AGT are adaptations that allow the enzyme to efficiently search for lesions in the context of chromatin remodeling and DNA replication 2.1.1.77 protein-L-isoaspartate(D-aspartate) O-methyltransferase molecular biology betaine administration of rats prevents the ethanol-induced accumulation of isoaspartyl-containing proteins in the liver by restoring the PIMT-catalyzed protein repair reaction through normalizing the hepatocellular SAM:SAH ratios 2.1.2.5 glutamate formimidoyltransferase molecular biology bifunctional formiminotransferase cyclodeaminase provides a novel marker to study ER-Golgi dynamics 2.2.1.2 transaldolase molecular biology TAL deficiency is shown as a modulator of mitochondrial homoeostasis, Ca2+ fluxing and apoptosis 2.2.1.6 acetolactate synthase molecular biology the G95A mutation of the ALS gene confers highly specific resistance to pyrimidinyl carboxy herbicides and can be used as a selection marker for transformations 2.2.1.6 acetolactate synthase molecular biology the gene is useful as a selectable marker for introducing foreign traits into rice when used with pyrimidinylcarboxylate herbicides. The double-mutant W548L/S627I of the ALS gene from rice is not only helpful for introducing useful rice genes into rice by self-cloning as a host-derived selectable marker gene but also can extinguish the scientific concern for antibiotic-resistant genes, leading to minimize public concern for this issue in transgenic plants 2.3.1.4 glucosamine-phosphate N-acetyltransferase molecular biology a microtiter plate based assay to detect the GlmU activity is developed: the assay relies on the enzymes MurA and MurB to convert UDP-GlcNAc into UDP-MurNAc with the concomitant reduction of NADPH. When all enzymes and substrates are present NADPH is oxidized with a concomitant decrease in OD340 2.3.1.4 glucosamine-phosphate N-acetyltransferase molecular biology an assay for glucosamine-6-phosphate synthase using a yeast glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) as coupling enzyme is developed. The assay measures the production of glucosamine-6-phosphate by either following the consumption of acetyl-CoA spectrophotometrically at 230 nm or quantifying the free thiol with 5,50-dithio-bis(2-nitrobenzoic acid) (Ellman’s reagent) in a discontinuous manner. This method is simple to perform and can be adapted to a 96-well microtiter plate format 2.3.1.5 arylamine N-acetyltransferase molecular biology results demonstrate that human P4501A1 and NATs (NAT1 and NAT2) contribute significantly to the activation of PBTA-type compounds to genotoxic metabolites that induce umuC gene expression in Salmonella typhimurium tester strains 2.3.1.5 arylamine N-acetyltransferase molecular biology results show that cisplatin inactivates the NAT1 enzyme by forming an adduct with the catalytic cysteine residue of the enzyme 2.3.1.12 dihydrolipoyllysine-residue acetyltransferase molecular biology dihydrolipoamide acetyltransferase is shown to be a metabolic longevity factor and is required for calorie restriction-mediated life span extension 2.3.1.B25 octaketide synthase molecular biology use of octaketide synthase for rational biosynthetic engineering to generate molecular diversity and pursue innovative, biologically potent compounds 2.3.1.28 chloramphenicol O-acetyltransferase molecular biology the chloramphenicol acetyl transferase gene serves as integration target for the eukaryotic mariner transposon Mos1 regardless of the location (chromosome or plasmid), as tested with in vitro and bacterial transposition assays 2.3.1.28 chloramphenicol O-acetyltransferase molecular biology single-chain variable fragment (scFv) phages are selected with affinity for CAT. Surface plasmon resonance analyses shows that the tested scFv phages have an affinity for CAT with a dissociation constant (Kd) around 1 microM. The selected scFv phages can be used as capture elements in a highly sensitive sandwich ELISA to detect CAT concentration as low as 0.1 ng/ml or 4 pM 2.3.1.28 chloramphenicol O-acetyltransferase molecular biology the enzyme mutant CATA138T may be useful as a genetic marker in Geobacillus spp. 2.3.1.30 serine O-acetyltransferase molecular biology results show that mitochondria provide the bulk of OAS in the plant cell and are the likely site of flux regulation 2.3.1.30 serine O-acetyltransferase molecular biology results show that some transgenic plants expressing serine acetyltransferase and cysteine synthase can mitigate detrimental effects of cadmium toxicity, perhaps by efficiently producing and accumulating sulfuric compounds 2.3.1.31 homoserine O-acetyltransferase molecular biology the HOA gene can be used as a selectable marker for transformation of Gibberella zeae 2.3.1.48 histone acetyltransferase molecular biology the catalytically inactive mutant Y891F is useful for stable binding and purification of unacetylated histone H4 protein 2.3.1.48 histone acetyltransferase molecular biology the results demonstrate the importance of TIPs in the recruitment of p300 to specific promoters and in the regulation of p300 HAT activity through the involvement of the SANT domain 2.3.1.50 serine C-palmitoyltransferase molecular biology results show that SPT modulated programmed cell death plays an important role in the regulation of male gametophyte development of Arabidopsis thaliana 2.3.1.97 glycylpeptide N-tetradecanoyltransferase molecular biology the enzyme can be used for protein N-myristoylation as a tag labeling technique in recombinant expresssion systems. CaNMT is an effective tool for in vitro and in vivo transfer of an azide-modified acid to the N-terminus of a polypeptide derived from a species entirely unrelated to Candida albicans 2.3.1.158 phospholipid:diacylglycerol acyltransferase molecular biology the conversion of a membrane bound lipid metabolizing enzyme into a soluble and active form might be applied to other enzymes with a membrane anchor region. 2.3.1.183 phosphinothricin acetyltransferase molecular biology the bar gene represents a selectable and assayable reporter gene especially suitable for 3’-terminal gene fusions 2.3.1.216 5,7-dihydroxy-2-methylchromone synthase molecular biology use of pentaketide chromone synthase for rational biosynthetic engineering to generate molecular diversity and pursue innovative, biologically potent compounds 2.4.1.41 polypeptide N-acetylgalactosaminyltransferase molecular biology enzyme will be useful for the in vitro glycosylation of proteins obtained from microorganisms by gene manipulation techniques 2.4.1.41 polypeptide N-acetylgalactosaminyltransferase molecular biology enzyme-linked lectin assay (ELLA) as carbohydrate-binding assay 2.4.1.41 polypeptide N-acetylgalactosaminyltransferase molecular biology invariant residue Trp328 is essential for GalNAc-T enzymatic activity, residue Trp316 is important in the interaction with the acceptor polypeptide of GalNAc-T1 2.4.1.41 polypeptide N-acetylgalactosaminyltransferase molecular biology overexpression in eukaryotic cell culture leads to inhibition of Activin/Nodal pathway activity in vivo by interference with binding of ActR-IIB to type I TGFbeta receptor proteins that can mediate BMP as well as Nodal signalling 2.4.1.41 polypeptide N-acetylgalactosaminyltransferase molecular biology polyclonal rabbit anti-GalNAc-T14 IgG (1: 1000 in Western blot, titer: appr. 1: 16000) for study of expression and distribution of human GalNAc-T14 2.4.1.65 3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase molecular biology the stable system using the expression vector pIB/Vf-His-TOPO constitutes an advance for the large scale expression of glycosyltransferases and possibly other glycoproteins in insect cells 2.4.1.80 ceramide glucosyltransferase molecular biology glucosylceramide is essential for MsDef1-mediated growth inhibition of Fusarium graminearum, but not for its pathogenicity 2.4.1.101 alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase molecular biology production of rare hybrid oligosaccharides for biochemical and structural studies, 100% conversion of oligosaccharide substrate at room temperature, yield of 42% after purification from reaction mixture 2.4.1.102 beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-N-acetylglucosaminyltransferase molecular biology beta-D-galactosyl-1,3-N-acetyl-D-galactosaminyl-p-nitrophenyl and other nitrophenyl-sugar-derivatives are useful as specific inhibitors and as affinity label 2.4.1.221 peptide-O-fucosyltransferase molecular biology engineering of an O-fucosylation system in yeast provides a powerful tool for producing proteins with homogenous carbohydrate chains. Such proteins can be used for the analysis of substrate specificity and the production of antibodies that recognize O-glycosylated EGF domains 2.4.1.221 peptide-O-fucosyltransferase molecular biology O-fucosylation is dispensable for many Notch signaling events during Drosophila development 2.4.2.8 hypoxanthine phosphoribosyltransferase molecular biology HPRT mutations in vivo in human T-lymphocytes are useful probes for mechanistic investigations, molecular analyses of isolated mutants reveal their underlying mutational changes as well as the T-cell receptor gene rearrangements present in the cells in question, overview 2.4.2.8 hypoxanthine phosphoribosyltransferase molecular biology the HPRT gene is used as reporter gene in HL-60 cells for investigation of the mutagenic potential of succinyl-acetone by determining the frequencies of somatic mutations in the HPRT reporter gene, overview 2.4.2.9 uracil phosphoribosyltransferase molecular biology engineering the yeast fluorocytosine deaminase (FCY1) gene by creating a fusion with the bacterial uracil phosphoribosyl transferase (UPP) gene results in a recombinant protein that converts the precursor 5-fluorocytosine (5-FC) into 5-fluorouracyl, a drug used in the treatment of a range of cancers, which triggers DNA and RNA damage. The tissue-specific FCY-UPP system is a great tool to inactivate cells in a precise spatial and temporal manner, method evaluation, overview 2.4.2.30 NAD+ ADP-ribosyltransferase molecular biology establishment of an immortalized PARP-1-/- murine endothelial cell line HYKO6 as a tool to study PARP-1-mediated endothelial cell dysfunction 2.5.1.10 (2E,6E)-farnesyl diphosphate synthase molecular biology farnesyl pyrophosphate synthase as a target for fragment-based lead discovery has revealed that it can be used for fragment library screening and hit validation using an unconventional referencing, suitable when reference compounds are not available 2.5.1.15 dihydropteroate synthase molecular biology established coupled enzymatic assay for kinetic analyses of DHPS activity (coupled to pyrophosphate-dependent phosphofructokinase, aldolase, triosephosphate isomerase, alpha-glycerophosphate dehydrogenase) in presence or absence of activity-modulating compounds 2.5.1.20 rubber cis-polyprenylcistransferase molecular biology model for rubber biosynthesis 2.5.1.59 protein geranylgeranyltransferase type I molecular biology the GGTase-I variants with altered substrate specificity can serve as tools for studying GGTase-I substrate selectivity and the effects of prenylation pathway modifications on specific proteins 2.5.1.78 6,7-dimethyl-8-ribityllumazine synthase molecular biology outside of the cell, the hollow spherical architecture of the enzyme capsid is used as a template for the encapsulation of cargo proteins, such as green fluorescent proteins, and HIV proteases, and fabrication of uniform layer-by-layer assemblies using non-covalent interactions between surface-displayed His6 and Ni-NTA of enzyme AaLS. The enzyme shows encapsulation capability and surface presentation of ligands, which represent the great potential of AaLS as a versatile delivery vehicle 2.6.1.19 4-aminobutyrate-2-oxoglutarate transaminase molecular biology Agrobacterium tumefaciens is used to mediate inter-kingdom DNA transfer in plant genetic engineering. Gamma-aminobutyric acid (GABA) is a negative factor in the Agrobacterium-plant interaction, because it inhibits the DNA transfer. Generation of an Agrobacterium tumefaciens strain expressing the Escherichia coli gene gabT, which introduces GABA transaminase activity and the ability to degrade GABA, is achieved to circumvent the inhibitory effect of GABA 2.7.1.21 thymidine kinase molecular biology a method to distinguish between the de novo induction of thymidine kinase mutants and the selection of pre-existing thymidine kinase mutants in the mouse lymphoma assay 2.7.1.60 N-acylmannosamine kinase molecular biology GNE-deficient cells, with dramatically increased incorporation of N-acetylmannosamine analogues into glycoproteins, can efficiently be decorated with reactive functional groups, which can be employed in bioorthogonal functionalization strategies for fluorescence labelling or biotinylation 2.7.1.119 hygromycin-B 7''-O-kinase molecular biology use the hygromycin phosphotransferase gene (hpt) as a selective marker gene for tracking plastid transformation in rice (Oryza sativa) 2.7.1.163 hygromycin B 4-O-kinase molecular biology the enzyme can be used as selective marker gene product in production of transgenic plants 2.7.1.163 hygromycin B 4-O-kinase molecular biology the mutant gene hph5 gene can be used as a selection marker in the host-vector system of Thermus thermophilus either on plasmid or by genome integration 2.7.1.163 hygromycin B 4-O-kinase molecular biology as selective marker gene 2.7.1.163 hygromycin B 4-O-kinase molecular biology gene is used as selectable marker, mediates hygromycin resistence 2.7.1.163 hygromycin B 4-O-kinase molecular biology gene sequence is used as selectable marker, mediates hygromycin resistance 2.7.1.163 hygromycin B 4-O-kinase molecular biology hpt gene is used as a selectable marker 2.7.1.163 hygromycin B 4-O-kinase molecular biology hpt gene is used as selectable marker, mediates hygromycin resistance 2.7.1.163 hygromycin B 4-O-kinase molecular biology resistance against hyromycin B mediated by transformation of the hph gene 2.7.1.163 hygromycin B 4-O-kinase molecular biology resistance against hyromycin B mediated by transformation of the hph gene, a selectable marker gen. 2.7.1.163 hygromycin B 4-O-kinase molecular biology selectable marker (SM) genes, essential to select transformed cells from a large population of untransformed cells 2.7.1.163 hygromycin B 4-O-kinase molecular biology Used as marker gene mediating hygromycin resistance. 2.7.1.163 hygromycin B 4-O-kinase molecular biology Used as marker gene. Mediates hygromycin resistance. 2.7.1.190 aminoglycoside 2''-phosphotransferase molecular biology there is a need to develop alternative markers for plastid transformation to (a) extend the species range of the technology, and (b) facilitate the multistep engineering of plastid genomes, for example, by sequential introduction of multiple transgenes (supertransformation). Bifunctional aminoglycoside acetyltransferase/phosphotransferase conferring tobramycin resistance provides an efficient selectable marker for stable plastid transformation 2.7.4.1 ATP-polyphosphate phosphotransferase molecular biology power of ppk1 as a genetic marker for detection of all currently defined Candidatus Accumulibacter clades 2.7.7.6 DNA-directed RNA polymerase molecular biology Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA 2.7.7.6 DNA-directed RNA polymerase molecular biology usage of enzyme RNAP to systematically decipher the black matter and identify giant viruses. As they lack ribosomal genes, these giant viruses are not part of the rDNA tree. A relevant alternative to rDNA for microbe identification are DNA-dependent RNA polymerase (RNAP) genes. They are more refractory to lateral gene transfers and usually present in a single copy in genomes, which avoids recombination and issues related to divergence between copies 2.7.7.7 DNA-directed DNA polymerase molecular biology thermostable polymerase used in PCR 2.7.7.7 DNA-directed DNA polymerase molecular biology generation of a unique one enzyme system with high fidelity to allow highly accurate and efficient amplification of DNA targets using polymerase chain reaction by fusing Sso7d protein to Tpa DNA polymerase 2.7.7.7 DNA-directed DNA polymerase molecular biology Tce polymerase may be useful in DNA amplification requiring high fidelity 2.7.7.7 DNA-directed DNA polymerase molecular biology the high fidelty of the enzyme is suitable fo polymerase chain reaction (PCR), which requires accurate DNA amplification for gene cloning and diagnostic assay 2.7.7.7 DNA-directed DNA polymerase molecular biology long and accurate PCR can be achieved with a mixture of wild type DNA polymerase from Thermococcus kodakaraensis and its exonuclease deficient mutant enzyme N210D is utilized (at the ratio of 1:40) 2.7.7.7 DNA-directed DNA polymerase molecular biology optimal conditions for polymerase chain reaction are determined. Iho DNA polymerase possesses 3'->5' exonuclease activity, and the fidelity of the Iho DNA polymerase is similar to that of Pfu and Vent DNA polymerases. However, Iho DNA polymerase provides more enhanced efficiency of PCR amplification than Pfu and Vent DNA polymerases. Iho DNA polymerase can successfully amplify a 2-kb lambda DNA target with a 10/s extension time and could amplify a DNA fragment up to 8 kb lambda DNA 2.7.7.7 DNA-directed DNA polymerase molecular biology the H633R mutant DNA polymerase may be useful in high-fidelity DNA amplification and various PCR-based applications 2.7.7.7 DNA-directed DNA polymerase molecular biology application for long and accurate PCR. The PCR error rate of the Tba5 DNA polymerase plus4 (Tba5 plus DNA polymerase mixtures are constituted with various amounts of Tba5 DNA polymerase mixed with Taq DNA polymerase) is much lower than that of the wild-type enzyme alone 2.7.7.7 DNA-directed DNA polymerase molecular biology the enzyme can be used in routine PCR 2.7.7.7 DNA-directed DNA polymerase molecular biology the thermostable properties of the enzyme from Thermus aquaticus have contributed majorly to the specificity, automation, and efficacy of the polymerase chain reaction (PCR) 2.7.7.8 polyribonucleotide nucleotidyltransferase molecular biology targeted overexpression of hPNPase represents a strategy to selectively downregulate RNA expression and consequently intervene in a variety of pathophysiological conditions 2.7.7.B22 transposase molecular biology Sleeping Beauty is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends 2.7.7.B22 transposase molecular biology the maize activator (Ac) transposase recognizes and excises Ac and Dissociation (Ds) elements and mediates insertion elsewhere in the genome. Insertions of Ds can cause disruption in gene sequences and hence are important functional genomics tool for tagging and cloning of unknown gene sequences 2.7.7.B22 transposase molecular biology transposases are important tools in genome engineering. The first DNA transposon tool capable for gene transfer in vertebrates is Sleeping Beauty (SB), which is reconstructed from extinct Tc1/mariner transposons in fish. Sleeping Beauty, and especially its hyperactive variant is still one of the most widely used transposon tools, in human clinical trials 2.7.7.48 RNA-directed RNA polymerase molecular biology the enzyme, that is absent in mammalian cells, enhances the effect of siRNA expression on target gene expression in mammalian cells, when coexpressed, for amplifying the use of hairpin siRNA expression vectors for gene silencing, method development, overview 2.7.7.64 UTP-monosaccharide-1-phosphate uridylyltransferase molecular biology polyclonal rabbit anti-AtUSP antibody recognizes USP1 and USP2 2.7.7.65 diguanylate cyclase molecular biology generation of structural models for product-inhibited, elongated dimer 2.7.8.7 holo-[acyl-carrier-protein] synthase molecular biology MtaA should be a usefool tool for activating heterologously expressed polyketide synthase and nonribosomal polyketide synthase systems 2.7.8.7 holo-[acyl-carrier-protein] synthase molecular biology insights in molecular architecture and reaction mechanism of group II PPTs in contrast to group I PPTs (bacterial) enable screening for antibacterial agents which specifically inhibit bacterial PPTs 2.7.8.7 holo-[acyl-carrier-protein] synthase molecular biology SchPPT is a promiscuous PPTase and may be used on polyketide production in heterologous bacterial host and labeling of acyl-carrier proteins, ACPs 2.7.8.13 phospho-N-acetylmuramoyl-pentapeptide-transferase molecular biology synthesis of radiolabelled UDP-MurNAc-pentapeptide as biochemical tools for studying peptidoglycan biosynthesis or the kinetic characterization of MraY 2.7.8.15 UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase molecular biology combination of GPT and tunicamycin is a potential selectable marker system for potato transformation, overview 2.7.11.1 non-specific serine/threonine protein kinase molecular biology AcMNPV-pk-1 is a component of the viral very late gene transcription initiation complex 2.7.11.1 non-specific serine/threonine protein kinase molecular biology EmbR2 is a regulator of PknH activation, thus directly participating in the control of the PknH/EmbR pair 2.7.11.1 non-specific serine/threonine protein kinase molecular biology pknA and pknB are key players in signal transduction pathways for the regulation of the cell shape and both are essential for sustaining corynebacterial growth 2.7.11.1 non-specific serine/threonine protein kinase molecular biology results demonstrate that MPK38 physically interacts with ASK1 in vivo and acts as a positive upstream regulator of ASK1 2.7.11.1 non-specific serine/threonine protein kinase molecular biology results display the requirement of RSK activity during Artemia development and suggest its role in termination of cell cycle (G2/Mphase) arrest and promotion of mitogenesis 2.7.11.1 non-specific serine/threonine protein kinase molecular biology SAPK4 regulates ion homeostasis and growth and development under salinity and this indicates a function of SAPK4 as a regulatory factor in plant salt stress acclimation 2.7.11.1 non-specific serine/threonine protein kinase molecular biology STK38 interacts with the MAPKKKs MEKK1 and MEKK2 (MEKK1/2). STK38 inhibits MEKK1/2 activation without preventing MEKK1/2 binding to its substrate, SEK1. STK38 suppresses the autophosphorylation of MEKK2 without interfering with MEKK2 dimer formation, and converts MEKK2 from its phosphorylated to its nonphosphorylated form. The negative regulation of MEKK1/2 is not due to its phosphorylation by STK38. STK38 negatively regulates the activation of MEKK1/2 by direct interaction with the catalytic domain of MEKK1/2, suggesting a novel mechanism of MEKK1/2 regulation 2.7.11.10 IkappaB kinase molecular biology IKK-alpha is a potential target for manipulating TLR-induced IFN-alpha production 2.7.11.10 IkappaB kinase molecular biology IKK/nuclear factor-kappaB dependent microglia activation contributes to KA-induced hippocampal neuronal cell death in vivo through induction of inflammatory mediators 2.7.11.10 IkappaB kinase molecular biology IKKalpha and IKKbeta exert differential roles in ECM remodeling and endochondral ossification, which are events characteristic of hypertrophic chondrocytes and also complicating factors often found in osteoarthritic chondrocytes 2.7.11.10 IkappaB kinase molecular biology it is shown that mTOR and Raptor, through an Akt-dependent pathway, control NF-kappaB activity in PC3 prostate cancer cells both at the level of IkappaBalpha and RelA/p65 phosphorylation 2.7.11.10 IkappaB kinase molecular biology NF-kappaB is activated by alloferon through down-regulation of antioxidant proteins and IkappaBalpha 2.7.11.10 IkappaB kinase molecular biology regulation of IKKbeta by miR-199a affects NF-kappaB activity in ovarian cancer cells 2.7.11.10 IkappaB kinase molecular biology tetrandrine suppresses LPS-induced astrocyte activation via modulating IKKs-IkappaBalpha-NF-kappaB signaling pathway 2.7.11.13 protein kinase C molecular biology a technique is developed to detect PKCalpha activity in a cancerous cell lysate through the simple measurement of fluorescence intensity. The principle of this methodology is based on a fluorescence increase associated with polyion complex dissociation due to phosphorylation by PKCalpha 2.8.2.20 protein-tyrosine sulfotransferase molecular biology conjugation of proteins with N-carbamoyl-succinate-modified peptides is an appropriate tool in research, for instance, in the development of vaccines and drugs or for studying biological mechanisms 2.8.3.8 acetate CoA-transferase molecular biology a new degenerated real-time PCR approach to simultaneously quantify phylogenetically different butyrate-producing bacteria based on the detection of butyryl-coenzyme A (CoA) CoA transferase genes is described 2.8.3.16 formyl-CoA transferase molecular biology use of the frc gene as template for PCR to detect oxalotrophic bacteria 3.1.1.6 acetylesterase molecular biology postulated PON (paraoxonase) family membership due to similarity to primary structure of PON2, plant strictosidine synthase and di-isopropyl fluorophosphatase, its N-terminal single transmembrane domain, a nine-exon gene structure, a six-bladed beta-propeller tertiary structure, and similar metabolic regulation of gene expression 3.1.1.6 acetylesterase molecular biology protein-protein interaction consensus sequence, involved in regulation of both sugar and lipid metabolism, according to interaction partners 3.1.1.17 gluconolactonase molecular biology coimmobilization with glucose oxidase in polyelectrolyte gels for improvement of kinetic properties, active enzymes in the gel undergo a shrinking process due to a sudden drop in pH, gel volume phase transition, overview 3.1.1.53 sialate O-acetylesterase molecular biology origin and evolution of viral hemagglutinin-esterases 3.1.1.56 methylumbelliferyl-acetate deacetylase molecular biology regulation of serine esterase by sulfenic acid found at C60 in mutant W197I (PDB: 3C6B) 3.1.1.56 methylumbelliferyl-acetate deacetylase molecular biology studying the physiological role 3.1.1.96 D-aminoacyl-tRNA deacylase molecular biology the dtd (yrvI) gene from B. amyloliquefaciensA50, encoding the putative metabolite proofreading enzyme D-tyrosyl-tRNATyr deacylase, is associated with resistance to the non-canonical amino acid D-tyrosine. This gene can be applied as a convenient, small selectable marker for non-antibiotic resistance selection in experiments aimed at genome editing of D-Tyr-sensitive microorganisms 3.1.1.117 (4-O-methyl)-D-glucuronate---lignin esterase molecular biology the enzyme may prove a valuable as research tool for the investigation of lignin and lignin to carbohydrates-bond chemistry 3.1.1.117 (4-O-methyl)-D-glucuronate---lignin esterase molecular biology the enzyme may prove a valuable research tool for the investigation of lignin and lignin to carbohydrates-bond chemistry 3.1.1.117 (4-O-methyl)-D-glucuronate---lignin esterase molecular biology the enzyme may prove valuable as a research tool for the investigation of lignin and lignin to carbohydrates-bond chemistry 3.1.3.1 alkaline phosphatase molecular biology analytically widely used enzyme, e.g. in ELISA, enzyme-linked immunosorbent assay 3.1.3.1 alkaline phosphatase molecular biology widely used in vitro, e.g. to dephosphorylate DNA or dNTPs, since the enzyme can be inactivated by a short rise in temperature 3.1.3.1 alkaline phosphatase molecular biology the purified alkaline phosphatase removes the 5'-phosphate group of a linearized plasmid without showing DNAase activity, indicating its potential for recombinant DNA technology 3.1.3.1 alkaline phosphatase molecular biology BAP may play an important role in differentiation and maturation of human B cells 3.1.3.1 alkaline phosphatase molecular biology colchicine inhibits the dexamethasone-promoted translocation of ALP to the plasma membrane surrounding the bile canaliculus-like structure in primary cultures of fetal rat hepatocytes by disassembling microtubules and discomposing the Golgi complex 3.1.3.1 alkaline phosphatase molecular biology influence of membrane lipid environment on the activity of GPI-anchored enzymes is investigated with PLAP reconstituted by a detergent-dialysis technique in liposomes composed of palmitoyloleoylphosphatidylcholine, alone or in mixture with lipids enriched along with the protein within lipid rafts. The highest Vmax is recorded for a phosphatidylcholine/10% monosialoganglioside, while the lowest for a phosphatidylcholine/30% cholesterol mixture and for raft-mimicking 1:1:1 phosphatidylcholine/sphingolipid/cholesterol liposomes. No significant differences in Km are detected. The GPI-enzyme activity is affected by membrane curvature 3.1.3.1 alkaline phosphatase molecular biology relationship between extracellular pH and the release of alkaline phosphatase using stimuli affecting extracellular pH, i.e. heat shock, nigericin, salicylicacid, benzoic acid, fusicoccin, NaOH, KOH, HCl, and the Golgi apparatus inhibitor BrefeldinA is investigated. Extracellular alkaline phosphatase is a sensitive marker for cellular stimulation 3.1.3.2 acid phosphatase molecular biology AcP and cysteine protease cooperate to assure vitellin breakdown during early embryogenesis of Periplaneta americana 3.1.3.2 acid phosphatase molecular biology ACP5 possesses a central role in removal of the mannose 6-phosphate recognition marker 3.1.3.2 acid phosphatase molecular biology APase activity may affect the tuber swelling by partially regulating the sucrose-mediated sugar resorption 3.1.3.2 acid phosphatase molecular biology the localization of an ACP in the arbuscular mycorrhizal (AM) interface of arbusculate coils suggests that this enzyme may be involved in the phosphorus efflux from themycorrhizal fungus to the host 3.1.3.2 acid phosphatase molecular biology TRAP activity is modulated during osteoblastic differentiation, possibly in response to the redox state of the cell 3.1.3.11 fructose-bisphosphatase molecular biology promoter methylation is involved in the regulation of expression of both FBPase isoenzymes in normal human tissues as well as in cancer 3.1.3.11 fructose-bisphosphatase molecular biology SEC28 (subunit of the coat protein complex I) is required for FBPase degradation. When SEC28 and other coatomer genes are mutated, FBPase degradation is defective and FBPase association with Vid vesicles is impaired 3.1.3.36 phosphoinositide 5-phosphatase molecular biology results raise the possibility that dynamic phosphoinositide metabolism may occur throughout the lifetime of a clathrin-coated pit 3.1.3.36 phosphoinositide 5-phosphatase molecular biology results suggest a role for INPP5B in retrograde ER-to-Golgi intermediate compartment (ERGIC) transport 3.1.3.36 phosphoinositide 5-phosphatase molecular biology SHIP2 lipid phosphatase activity plays an important role in the metabolism of PtdIns(3,4,5)P3 in response to H2O2 3.1.3.36 phosphoinositide 5-phosphatase molecular biology the interaction between adaptor protein APS and SHIP2 provides to both proteins potential negative regulatory mechanisms to act on the insulin cascade 3.1.3.36 phosphoinositide 5-phosphatase molecular biology translocatable phosphatase: method is based on the rapamycin-induced heterodimerization of FRB (fragment of mammalian target of rapamycin that binds FKB12) and FKB12 (FK506 binding protein 12). A mutant version of 5-ptase with a defective membrane targeting domain (CAAX box) is fused to FKB12 and tagged with monomeric red fluorescent protein, whereas its binding partner FRB (fused to CFP) is tethered to the plasma membrane through palmitoylation. In the absence of rapamycin, 5-ptase resides in the cytosol and leaves D-myo-phosphatidylinositol 4,5-bisphosphate levels at the plasma membrane unaltered. Upon addition of 100 nM rapamycin, FKB12 and FRB undergo heterodimerization and the 5-ptase is recruited to the plasma membrane 3.1.3.36 phosphoinositide 5-phosphatase molecular biology c-Jun NH2-terminal kinase (JNK)-interacting protein 1 (JIP1) interacts with SHIP2 and thereby positively modulates the MLK3/JIP1-mediated JNK1 activation. Furthermore, SHIP2 positively regulates the tyrosine phosphorylation of JIP1. By its interacting properties, SHIP2 can modulate JIP1-mediated JNK pathway signaling 3.1.3.36 phosphoinositide 5-phosphatase molecular biology catalytically inactive SHIP2 mutant P686A/D690A/R691A reduces preadipocyte proliferation by attenuating PDGFR signaling 3.1.3.36 phosphoinositide 5-phosphatase molecular biology hydrolysis of phosphatidylinositol 4,5-bisphosphate by PI(4,5)P2 5-phosphatase mediates calcium-induced inactivation of TRPV6 channels 3.1.3.36 phosphoinositide 5-phosphatase molecular biology intersectin1 (ITSN1) is identified as a binding partner of the SH2 domain containing inositol 5-phosphatase 2 (SHIP2). In response to epidermal growth factor, SHIP2 expression is able to recruit the ITSN1 short form (ITSN1-S) to the cell membrane, while SHIP2 overexpression does not modulate the ITSN-mediated extracellular signal-regulated kinase1/2 and c-Jun NH2-terminal kinase activation 3.1.3.36 phosphoinositide 5-phosphatase molecular biology OCRL1 exists as two functional pools, one participating in clathrin-mediated trafficking events such as endocytosis, and another that is much less or not involved in this process 3.1.3.36 phosphoinositide 5-phosphatase molecular biology SKIP is translocated from endoplasmic reticulum into nucleus through its interaction with hepatitis B virus core protein and suppresses HBV gene expression via a novel suppression domain 3.1.3.43 [pyruvate dehydrogenase (acetyl-transferring)]-phosphatase molecular biology pyruvate dehydrogenase complex in yeast is regulated by the PDH phosphatase Yor090cp 3.1.3.43 [pyruvate dehydrogenase (acetyl-transferring)]-phosphatase molecular biology pyruvate dehydrogenase phosphatase acts as a Smad phosphatase and plays a key role in decapentaplegic signalling pathways, reduced Smad1 dephosphorylation after neutralisation of PDP1 or PDP2 with SiRNA 3.1.3.43 [pyruvate dehydrogenase (acetyl-transferring)]-phosphatase molecular biology pyruvate dehydrogenase phosphatase acts as a Smad1 phosphatase and plays a key role Decapentaplegic signalling pathways 3.1.3.43 [pyruvate dehydrogenase (acetyl-transferring)]-phosphatase molecular biology pyruvate dehydrogenase phosphatase acts as a Smad1 phosphatase and plays a key role in TGF-b signalling pathways 3.1.3.43 [pyruvate dehydrogenase (acetyl-transferring)]-phosphatase molecular biology pyruvate dehydrogenase phosphatase acts as a Smad1 phosphatase and plays a key role TGF-b signalling pathways 3.1.3.46 fructose-2,6-bisphosphate 2-phosphatase molecular biology overexpression alone has negligible effects on insulin secretion, only in combination with 6-phosphofructo-2-kinase, partial down-regulation of endogenous PFKFB2 and PFKFB3 in INS1 cells by siRNA decreased PFK-2/FBPase-2 protein, fructose 2,6-bisphosphate content, glucokinase activity and glucose-induced insulin secretion 3.1.3.56 inositol-polyphosphate 5-phosphatase molecular biology At5PTase1 and At5PTase2 genes have nonredundant roles in hydrolyzing inositol second-messenger substrates and regulation of Ins(1,4,5)P3 levels is important during germination and early seedling development 3.1.3.57 inositol-1,4-bisphosphate 1-phosphatase molecular biology contains a D-domain as mitogen-activated protein kinase docking site but no FXFP motif 3.1.3.57 inositol-1,4-bisphosphate 1-phosphatase molecular biology contains no D-domain and no FXFP motif as mitogen-activated protein kinase docking site 3.1.4.4 phospholipase D molecular biology Ca2+-induced generation of membrane microdomains dramatically activates alpha-type phospholipase D from white cabbage 3.1.4.4 phospholipase D molecular biology functional roles of CYP1A2 and CYP2E1 can be switched by interaction with a specific anionic lysophospholipid in vivo 3.1.4.4 phospholipase D molecular biology knockout and overexpression of PLDalpha3 alter plant response to salinity and water deficit. Alterations of PLDalpha3 result in changes in phosphatidic acid level and membrane lipid composition. PLDalpha3-knockout plants display increased sensitivities to salinity and water deficiency and also tend to induce abscisic acid-responsive genes more readily than wild-type plants, whereas PLDalpha3-overexpressed plants have decreased sensitivities. PLDalpha3-knockout plants flower later than wild-type plants in slightly dry conditions, whereas PLDalpha3-overexpressed plants flower earlier 3.1.4.4 phospholipase D molecular biology phospholipase C and PLD signaling pathways are involved in the transduction and/or perception of the inductive stimulus that is emitted by the guard mother cells and induces the polar actin filament organization and asymmetric PLC and PLD signaling pathways are involved in the transduction and/or perception of the inductive stimulus that is emitted by the guard cell mothers and induces the polar actin filament organization and asymmetric subsidiary cell mother cell division 3.1.4.4 phospholipase D molecular biology PLD may differentially play a role in the course of early development of the brain, with special reference to the cerebrum and hindbrain, in rats 3.1.4.4 phospholipase D molecular biology PLD may differentially play a role in the course of early development of the brain, with special reference to the cerebrum and hindbrain,in rats 3.1.4.4 phospholipase D molecular biology PLD product phosphatidic acid acts as a membrane anchor of Rac1. The C-terminal polybasic motif of Rac1 is responsible for direct interaction with phosphatidic acid. It is shown that phosphatidic acid induces dissociation of Rho-guanine nucleotide dissociation inhibitor from Rac1 and that phosphatidic acid-mediated Rac1 localization is important for integrin-mediated lamellipodia formation, cell spreading, and migration 3.1.4.4 phospholipase D molecular biology PLD1 and not PLD2 is specifically required for regulated secretion of von Willebrand factor from endothelial cells 3.1.4.4 phospholipase D molecular biology the critical role of PLD1 in the intracellular signaling cascades initiated by TNF-alpha and its functional role for coordinating the signals to inflammatory responses 3.1.4.17 3',5'-cyclic-nucleotide phosphodiesterase molecular biology assay procedure of calmodulin-dependent cyclic nucleotide phosphodiesterase 3.1.4.17 3',5'-cyclic-nucleotide phosphodiesterase molecular biology convenient and sensitive radioenzymatic assay for characterization and determining the contribution if the various PDE families in cell and tissue, PDE11 3.1.4.17 3',5'-cyclic-nucleotide phosphodiesterase molecular biology convenient and sensitive radioenzymatic assay for characterization and determining the contribution of the various PDE families in cell and tissue, PDE1 3.1.4.17 3',5'-cyclic-nucleotide phosphodiesterase molecular biology convenient and sensitive radioenzymatic assay for characterization and determining the contribution of the various PDE families in cell and tissue, PDE10 3.1.4.17 3',5'-cyclic-nucleotide phosphodiesterase molecular biology convenient and sensitive radioenzymatic assay for characterization and determining the contribution of the various PDE families in cell and tissue, PDE2 3.1.4.17 3',5'-cyclic-nucleotide phosphodiesterase molecular biology convenient and sensitive radioenzymatic assay for characterization and determining the contribution of the various PDE families in cell and tissue, PDE3 3.1.4.17 3',5'-cyclic-nucleotide phosphodiesterase molecular biology description of a technique used to estimate the extent of PDE1 activation in vivo by measuring in vitro the PDE activity. The technique can be used to measure Ca2+/CaM-stimulated PDE activity in cultured cells or tissues 3.1.4.35 3',5'-cyclic-GMP phosphodiesterase molecular biology convenient and sensitive radioenzymatic assay for characterization and determining the contribution if the various PDE families in cell and tissue, PDE5 3.1.4.35 3',5'-cyclic-GMP phosphodiesterase molecular biology convenient and sensitive radioenzymatic assay for characterization and determining the contribution if the various PDE families in cell and tissue, PDE6 3.1.4.35 3',5'-cyclic-GMP phosphodiesterase molecular biology convenient and sensitive radioenzymatic assay for characterization and determining the contribution if the various PDE families in cell and tissue, PDE9 3.1.4.39 alkylglycerophosphoethanolamine phosphodiesterase molecular biology is involved in the regulation of IL-8 and CD54 gene 3.1.4.53 3',5'-cyclic-AMP phosphodiesterase molecular biology convenient and sensitive radioenzymatic assay for characterization and determining the contribution if the various PDE families in cell and tissue, PDE4 3.1.4.53 3',5'-cyclic-AMP phosphodiesterase molecular biology convenient and sensitive radioenzymatic assay for characterization and determining the contribution if the various PDE families in cell and tissue, PDE7 3.1.4.53 3',5'-cyclic-AMP phosphodiesterase molecular biology convenient and sensitive radioenzymatic assay for characterization and determining the contribution if the various PDE families in cell and tissue, PDE8 3.1.4.53 3',5'-cyclic-AMP phosphodiesterase molecular biology development of cell-permeable peptide reagents based upon the N-terminal region of PDE4D5 that allow for the selective disruption of PDE4D5 targeting to specific signalling scaffolds, namely beta-arrestin and RACK1 3.1.6.8 cerebroside-sulfatase molecular biology Ars can be very useful for clarifying the mechanisms underpinning syndromes caused by the deficiency of the function of Ars genes 3.1.11.1 exodeoxyribonuclease I molecular biology Exo1 plays an important role in the induction of apoptosis by unrepaired O6-methylguanines 3.1.11.2 exodeoxyribonuclease III molecular biology in contrast to DNA cloning utilizing in vitro recombination, some strains of Escherichia coli can take up linear double-stranded vectors, insert DNA fragments, and assemble them in vivo involving exonulcease III. The ends of these linear DNA fragments must contain 20 to 50 bp of overlapping homologous sequences. Improved protocols for in vivo cloning have realized a high level of usability comparable to that by in vitro recombination reactions, but using the exonuclease III, it is only necessary to introduce PCR products into Escherichia coli for the in vivo cloning 3.1.11.3 exodeoxyribonuclease (lambda-induced) molecular biology nascent strand sequencing (NS-seq) is used to discover DNA replication origins genome-wide, allowing identification of features for their specification. NS-seq depends on the ability of lambda exonuclease (lambda-exo) to efficiently digest parental DNA while leaving RNA-primer protected nascent strands intact. LexoG0 data to control for nascent strand-independent lambda-exo biases in NSseq and validated this approach at the rDNA locus. Possible increase of both sensitivity and specificity of NS-seq, overview 3.1.15.1 venom exonuclease molecular biology the enzyme is useful for production of the mass ladders of oligodeoxynucleotides with a single nucleotide difference, which can be produced, allowing for MS-based sequencing, construction of a capillary monolithic bioreactor constructed for the immobilized enzyme and handling (sub)microliter DNA samples 3.1.21.1 deoxyribonuclease I molecular biology a DNase bioreactor can be used to remove DNA from RNA samples prior to reverse transcription followed by PCR 3.1.21.1 deoxyribonuclease I molecular biology DNase-I hypersensitive sites may constitute a molecular marker to identify alleles and subsequently acquire the appropriate methylation imprint. This molecular identifier may be present or absent for a specific gene according to the sex of the gamete 3.1.21.B3 mismatch-specific endonuclease molecular biology the thermostable mismatch-specific endonuclease will be applicable to genetic engineering techniques 3.1.21.4 type II site-specific deoxyribonuclease molecular biology type II REases are widely used as tools for the dissection, analysis and reconstruction of DNA 3.1.21.4 type II site-specific deoxyribonuclease molecular biology tools for the dissection, analysis and reconstruction of DNA 3.1.21.4 type II site-specific deoxyribonuclease molecular biology a straightforward, general and automatable model system for studying the activity of restriction endonucleases by using massively parallel sequencing is described, which should be highly applicable for future studies of large sets of restriction endonucleases and their activity 3.1.21.4 type II site-specific deoxyribonuclease molecular biology a straightforward, general and automatable model system for studying the activity of restriction endonucleases by using massively parallel sequencing is described, which should be highly applicable for the future studies of large sets of restriction endonucleases and their activity 3.1.21.4 type II site-specific deoxyribonuclease molecular biology protein tagging with a wide variety of epitopes and/or fusion partners is used routinely to dissect protein function molecularly. Frequently the required DNA subcloning is inefficient, especially in cases where multiple constructs are desired for a given protein with unique tags. The generated clones have unwanted junction sequences introduced. To add versatile tags into the extracellular domain of the transmembrane protein THSD1, a protein tagging technique is developed that utilizes non-classical type IIS restriction enzymes that recognize non-palindromic DNA sequences and cleave outside of their recognition sites. The method is highly efficient and can precisely fuse any tag into any position of a protein in a scarless manner. IT is cost-efficient and adaptable because it uses commercially available type IIS restriction enzymes and is compatible with the traditional cloning system used by many labs 3.1.21.4 type II site-specific deoxyribonuclease molecular biology site-directed mutagenesis methods are very important in modern molecular biology, biochemistry, and protein engineering. A site-directed mutagenesis method that can be used for multiple mutation generation using type IIs restriction enzymes. This approach is faster and more convenient than the overlap polymerase chain reaction method due to its having fewer reaction steps and being cheaper than, but as convenient as, enzymatic assembly 3.1.21.7 deoxyribonuclease V molecular biology application to DNA shuffling. Random DNA fragmentation by endonuclease V is a handy and reproducible method and can be used instead of fragmentation by DNase I, which is technically problematic due to decreasing shuffling efficiency 3.1.25.1 deoxyribonuclease (pyrimidine dimer) molecular biology UV-induced cell damage in xeroderma pigmentosa cells can be repaired by properly delivered T4 endonuclease V and can be used as a model for investigation of DNA repair, mutagenesis, and cell signaling without manipulation of the genome 3.1.25.1 deoxyribonuclease (pyrimidine dimer) molecular biology the enzyme is used as a gold standard for detection and/or calibration of DNA lesions (T4 endonuclease V-sensitive sites) caused by UVA damage 3.1.26.4 ribonuclease H molecular biology the enzyme can be useful in regulation of RNA degradation in applications of technologies involving mRNA, method development, overview 3.1.30.1 Aspergillus nuclease S1 molecular biology mutation detection in DNA oligonucleotides based on a guanine-quenching method coupled with T7 endonuclease-catalysed digestion of single-stranded DNA 3.1.30.1 Aspergillus nuclease S1 molecular biology straightforward detection of single-nucleotide polymorphisms in double-stranded DNA by using exonuclease III/nuclease S1/peptide nucleic acid system 3.1.30.1 Aspergillus nuclease S1 molecular biology a combination of S1 nuclease with two strands of pseudo-complementary peptide nucleic acid is useful for cleaving genomic DNA into desired fragments at targeted sites. The DNA analogue pseudo-complementary peptide nucleic acid, pcPNA, has a poly[N-aminoethylglycine] backbone and invades double-stranded DNA through Watson-Crick base-pairing. Through the invasion, single-stranded portions are formed at targeted sites in the genome and cut by the enzyme which hydrolyses only single-stranded DNA. The enzyme's mismatch-recognition activity was high enough to distinguish one base-pair difference in the invasion site. Enzyme cofactor Zn2+ suppresses the DNA invasion by pseudo-complementary peptide nucleic acid 3.1.30.2 Serratia marcescens nuclease molecular biology the enzyme is useful in DNA enrichment by PCR based applications for which removal of template DNA is beneficial before end repair, method overview. Mung bean nuclease treatment abolished the high molecular DNA smear in microdroplet-PCR captured DNA after end repair 3.1.31.1 micrococcal nuclease molecular biology purified enzyme can be used as an exogenous reagent to clear cellular extracts and improve protein purification 3.1.31.1 micrococcal nuclease molecular biology the enzyme is a useful tool for mapping chromatin structure in eukaryotes, usage of the enzyme to determine the positions of nucleosomes within a region of DNA to identify dynamic changes induced during gene regulation 3.1.31.1 micrococcal nuclease molecular biology combined MNase/exoIII digestion can be applied to in situ chromatin for unbiased genome-wide mapping of nucleosome positions that is not influenced by DNA sequences at the core/linker junctions. The same approach can be also used for the precise mapping of the extent of linker DNA protection by H1 and other protein factors associated with nucleosome linkers 3.2.1.2 beta-amylase molecular biology usage of beta-amylase phylogenetic signal to reconstruct the evolutionary history of the Pooid grasses. Phylogenetic relationships of 37 grass species (26% of the sampled species) highlight the frequent occurrence of non-treelike evolutionary events, so this LCNG should be used with caution as a phylogenetic marker 3.2.1.14 chitinase molecular biology recombinant Lpp-OmpA-ChBD fusion protein is useful for stable and highly specific immobilization of cells, displaying the fusion protein on the cell surface, on chitinous material, method optimization, overview 3.2.1.14 chitinase molecular biology the chitin-binding domain ChBD of Bacillus circulans strain WL-12 chitinase A1 is useful in the immobilization of D-hydantoinase on chitin beads for usage as long-life biocatalyst with high tolerance for heat, overview 3.2.1.14 chitinase molecular biology the polymorphic gene encoding the enzyme is a marker for species determination in Entamoeba 3.2.1.18 exo-alpha-sialidase molecular biology use of the enzyme in the desialylation of human recombinant erythropoietin 3.2.1.21 beta-glucosidase molecular biology a reporter gene for the localization of mammalian cells and transgenic tissues based on detection of the bglA (SYNbglA) gene of Caldocellum saccharolyticum that encodes a thermophilic beta-glucosidase is presented. SYNbglA expression can be localized in situ or detected quantitatively in colorimetric assays and can be co-localized with Escherichia coli beta-galactosidase. SYNbglA can be detected in tissue wholemounts and in frozen and wax embedded sections 3.2.1.22 alpha-galactosidase molecular biology alpha-galactosidase is used as a simple yet powerful reporter enzyme system in Saccharopolyspora erythraea. This reporter sytem has the distinct advantage of quantitatively measuring the level of alpha-galactosidase activity quickly and easily from culture supernatant 3.2.1.23 beta-galactosidase molecular biology senescence-associated beta-galactosidase activity is a widely used biomarker for assessing replicative sensescence in mammalian cells. Quantitative assay of senescence-associated beta-galactosidase activity in mammalian cell extracts. The assay is capable of detecting relatively subtle changes in activity and confirms that confluency and contact inhibition of growth can cause a small increase in the expression of this biomarker. The assay for measuring senescence-associated changes in beta-galactosidase is suitable for mechanistic studies of senescence regulation in which graduated changes in biomarker expression may be anticipated 3.2.1.23 beta-galactosidase molecular biology expression of bgaH under the control of various halobacterial promoters of known strength leads to different specific beta-galactosidase activities in the lysates. Using Northern blot hybridization and semiquantitative RT-PCR, it is shown that the bgaH transcript level corresponds to the specific enzyme activity. Therefore, the bgaH gene of Haloferax alicantei is a useful tool for in vivo studies of gene expression in Halobacterium salinarum and possibly other halophilic Archaea 3.2.1.31 beta-glucuronidase molecular biology stable expression of GusA in Gardia lamblia strain WB C6 for establishing a system to test enzyme susceptibility to the anti-giardial drugs nitazoxanide and metronidazole, overview 3.2.1.31 beta-glucuronidase molecular biology beta-glucuronidase is the most frequent reporter gene in plants. Beta-glucuronidase enzyme activity is not only tissue-specific but also genotype-dependent 3.2.1.31 beta-glucuronidase molecular biology use of beta-glucuronidase from Thermotoga maritima as a thermostable marker in higher plants, reporter enzyme in plant genetic research 3.2.1.35 hyaluronoglucosaminidase molecular biology the influence of hyaluronidase digestion modulates CD44 expression in various cancer cell lines 3.2.1.B43 keratanase II molecular biology truncated forms of keratanase II exhibit activity and excellent storage and thermal stability making these useful tools for glycobiology research 3.2.1.83 kappa-carrageenase molecular biology potential use in the future in the field of seaweed biotechnology for obtaining protoplasts, and in molecular biology to prevent severe separation problems occuring in the presence of phycocolloid oligomers 3.2.1.129 endo-alpha-sialidase molecular biology removal of target molecules to investigate their function 3.2.1.139 alpha-glucuronidase molecular biology screening instrument to find alpha-glucuronidase genes in DNA libraries in solid phase that enables higher throughput compared to liquid phase assays - screening of 50000 clones per 15-cm petri dish, addition of beta-xylosidase increases signal, the enzyme is important in facilitating the cellulose breakdown for biofuel production 3.2.1.143 poly(ADP-ribose) glycohydrolase molecular biology specific killing of DNA damage-response deficient cells with inhibitors of poly(ADP-ribose) glycohydrolase 3.2.1.146 beta-galactofuranosidase molecular biology labeled substrate to detect galactofuranosidase activity, galactofuranose metabolism is a potential target for chemotherapeutic agents to fight microbial infections 3.2.2.27 uracil-DNA glycosylase molecular biology UDG forms an apyrimidinic site, where an uracil base is removed, and facilitates the reactions of various DNA-repairing enzymes, such as AP endonuclease, DNA polymerase, and DNA ligase, it is thus useful in applications to remove uracil-DNA. Use of UDG to control carry-over contamination in PCR 3.4.14.9 tripeptidyl-peptidase I molecular biology fluorescent method for the histochemical detection of tripeptidyl peptidase I using glycyl-L-prolyl-L-Met-2-anthraquinonyl hydrazide as substrate 3.4.16.5 carboxypeptidase C molecular biology a method is described exploiting the possibility to attach different reactive handles to their C-termini using a reaction catalyzed by CPY. It is possible to attach pairs of reaction handles which can react with each other to each of the peptides to be coupled. In a second step, the two modified peptides can be linked together by a chemical reaction, such as an oxime-forming reaction or a copper(I) catalyzed (2+3)-cycloaddition reaction of an azide with an alkyne 3.4.16.6 carboxypeptidase D molecular biology Kex2 and Kex1 may promote cell fusion by proteolytically processing substrates that act in parallel to Prm1 (pheromone-regulated membrane protein 1) as an alternative fusion machine, as cell wall components, or both 3.4.17.20 Carboxypeptidase U molecular biology results demonstrate that the activation peptide is not required for TAFIa activity or for stabilization of the enzyme, but solely for stabilization of the zymogen 3.4.17.20 Carboxypeptidase U molecular biology thrombomodulin suppresses pericellular fibrinolysis and plasma-induced tumor cell invasion which is mediated by plasma TAFI activation 3.4.19.12 ubiquitinyl hydrolase 1 molecular biology Pichia pastoris is a robust system to express the secreted form of Drosophila melanogaster UCH 3.4.21.9 enteropeptidase molecular biology the enzyme is used for cleavage of the N-terminal part of recombinant human interferon-alpha2a (IFN-alpha2a) and IFN-alpha2b, expressed in Escherichia coli strains strains BL21 and BL21 (DE3), for production of the protein without the N-terminal methionine residue 3.4.21.9 enteropeptidase molecular biology the high degree of specificity exhibited by enteropeptidase makes it a suitable reagent for cleaving recombinant proteins to remove affinity or other tags. However often unwanted cleavages elsewhere in the protein occur during cleavage of fusions when high amount of enzyme is required 3.4.21.9 enteropeptidase molecular biology the high specificity of the target site makes enterokinase an ideal tool for cleaving fusion proteins at defined cleavage sites 3.4.21.61 Kexin molecular biology role of Kex2 during cell fusion. Kex2 may promote cell fusion by proteolytically processing substrates that act in parallel to Prm1 as an alternative fusion machine, as cell wall components, or both 3.4.21.64 peptidase K molecular biology proteinase K from Tritirachium album, which is one of the most widely used proteases in molecular biological studies. The synthesized linear oligo-phenylalanine shows a unique self-assembly in aqueous solutions 3.4.21.86 Limulus clotting enzyme molecular biology laser scattering photometry can be used to measure the formation of small particles of clotted enzyme, the timing is related to endotoxin concentration, this method can be used for quick and sensitive endotoxin assay 3.4.21.91 Flavivirin molecular biology it is demonstrated that the WNV NS2B/NS3pro-hel is an active endoproteolytic enzyme at the ER membrane and the in-cell selectivity profiling of the membrane-anchored flaviviral protease is reported 3.4.21.94 proprotein convertase 2 molecular biology the regulation of the prohormone processing system by morphine may lead to alterations in the levels of multiple bioactive hormones and may be a compensatory mechanism whereby the organism tries to restore its homeostatic hormonal milieu. The down-regulation of PC1/3, PC2 and P-CREB by short-term morphine and up-regulation by long-term morphine treatment may be a signal mediating the switch from drug use to drug abuse 3.4.21.98 hepacivirin molecular biology in order to develop an efficient screening method to identify viral proteins and their ability to block Jak-Stat signaling, the 2FTGH (human fibrosarcoma cells) cell assay system is used in combination with transient transfection of hepatitis C virus proteins: Using 1000 U/ml interferon and 30 mM 6-thioguanine to treat 2FTGH cells, it is established that transient protein expression in this cell system yields 39% and 0% cell survival for the positive (HPV E7) and negative controls (GFP expression) respectively. Transient expression of HCV Core-p7 results in 22% cell survival, consistent with previous reports, while expression of the HCV serine protease NS3/4a results in 54% cell survival. Furthermore, it is shown that NS3/4a inhibits phosphorylation of Stat-1 at the serine residue 3.4.21.104 mannan-binding lectin-associated serine protease-2 molecular biology near monodisperse endotoxin-free polyethylene glycol, at concentrations relevant to therapeutic effects, trigger complement activation in human sera. Depending on polyethylene glycol concentration and average molecular weight, complement activation proceeds either exclusively through MASP-2 activation, and a likely role for the lectin pathway, or through both MASP-2-mediated C4 cleavage and accelerated alternative pathway turnover 3.4.21.105 rhomboid protease molecular biology TM5 helix and L1 loop are dynamically coupled so that changes in the dynamics of one are relayed to the other 3.4.21.107 peptidase Do molecular biology htrA gene in Porphyromonas gingivalis does not relate to stress conditions such as high temperature and pH, but rather to H2O2 stress. The htrA gene is important for virulence and survival in in vivo animal models 3.4.21.107 peptidase Do molecular biology instead of using an Lactococcus lactis htrA mutant, the reduction of the HtrA level in wild-type recombinant cultures of Lactococcus lactis by acid tolerace response (ATR) suppression may serve as a better strategy for the production of secreted recombinant proteins 3.4.21.117 stratum corneum chymotryptic enzyme molecular biology an activity assay for human kallikrein 7 is developed using a blue-fluorescent acridone dye, featuring a remarkably long lifetime that can be quenched by either of the 2 natural amino acids, tyrosine and tryptophan. Incorporating this probe and 1 of the quenching amino acids on either side of the scissile bond of the substrate peptide makes it possible to monitor the enzymatic activity by quantifying the increase in the fluorescence lifetime signal. A systematic investigation of substrate structures leads to a homogenous, microplate-based, compound profiling assay that yields inhibitory constants down into the single-digit nanomolar range 3.4.21.117 stratum corneum chymotryptic enzyme molecular biology kallikrein 7 and antileukoprotease are down-regulated in prostate cancers 3.4.21.117 stratum corneum chymotryptic enzyme molecular biology kallikrein 7 may play a critical role in the invasion of cancer cells in which it is aberrantly expressed by degrading important cell adhesive molecules like desmogleins and E-cadherin 3.4.21.118 kallikrein 8 molecular biology miRNAs play a role in the regulation of KLK expression: using a bioinformatics approach 96 strong KLK/miRNA interactions are identified. KLK10 is the most frequently targeted kallikrein, followed by KLK5 and KLK13. KLK1, KLK3, KLK8 and KLK12 do not have strongly predicted miRNA/KLK interactions 3.4.21.122 transmembrane protease serine 2 molecular biology establishement of MDCK cell lines stably expressing TMPRSS2. The cell line stably expresses TMPRSS2 after 20 serial passages 3.4.22.1 cathepsin B molecular biology N,N'-diBoc-dityrosine-Gly-(isoniazid)2 is a sensitive and selective assay of cathepsin B activity 3.4.22.14 actinidain molecular biology actinidin compared with type II or IV collagenase isolates intact human umbilical vein endothelial cells, hepatocytes, and thymic epithelial cells with viability more than 90% 3.4.22.28 picornain 3C molecular biology a protein-tagging system for purification of EGFP from Escherichia coli using a single-step glutathione column purification and release of EGFP from the column using a variant of Human Rhinovirus 14 3C is described. A biotinylated variant of Human Rhinovirus 14 3C protease (bHR3Cp) is constructed, which includes a factor Xa or thrombin cleavage site between the protease gene and the biotinylation signal. This system readily allows attachment of the protease to a variety of surfaces and subsequent release using either thrombin or factor Xa proteases 3.4.22.28 picornain 3C molecular biology human rhinovirus (HRV) 3C protease is widely used in recombinant protein production for various applications such as biochemical characterization and structural biology projects to separate recombinant fusion proteins from their affinity tags in order to prevent interference between these tags and the target proteins 3.4.22.29 picornain 2A molecular biology picornaviral 2A sequences can be used to express transgenes in oncolytic adenoviruses 3.4.22.37 gingipain R molecular biology enzyme is a convenient tool for protein chemistry due to its stability and activity under conditions of high detergent concentration used in protein solubilization and purification 3.4.22.47 gingipain K molecular biology a combination of both R- and K-gingipains is required for pigment production from oxyhemoglobin by Porphyromonas gingivalis since R-gingipain converts oxyhemoglobin into the methemoglobin form which is more susceptible to Kgp degradation for the eventual release of iron(III) protoporphyrin IX and production of the micro-oxo haem dimer 3.4.22.47 gingipain K molecular biology it is shown that the production of anionic polysaccharide at the surface of Porphyromonas gingivalis rather than Arg- and Lys-gingipain synthesis is the principal mechanism of serum resistance in Porphyromonas gingivalis 3.4.22.49 separase molecular biology data suggest that the co-ordinated expression of separase, securin and Rad21 is fundamental for the developing brain 3.4.22.49 separase molecular biology in Saccharomyces cerevisiae separase it is shown that separase is implicated in a second non-proteolytic pathway: separsae is essential for the activation of Cdc14 phosphatase and thus a broad programme of late mitotic events culminating in mitotic exit and cell division 3.4.22.49 separase molecular biology separase is identified as a key cell cycle component that is required for degranulation 3.4.22.49 separase molecular biology separse is not only required for chromosome segregation but also for meiotic exit 3.4.22.49 separase molecular biology the results indicate that inhibitory phosphorylation of separase plays a critical role in the maintenance of sister chromatid cohesion and genome stability in proliferating postmigratory primordial germ cells 3.4.22.49 separase molecular biology the results show that a fraction of arm cohesin is protected by Sgo1, which prevents cohesin from being removed by the prophase pathway, and that separase is partly activated in nocodazole-arrested cells and removes the arm cohesion protected by Sgo1 3.4.22.51 cruzipain molecular biology in this study it is shown in murine macrophage cell line J774 as well as in murine bone marrow-derived macrophages, that cruzipain induces p38 phosphorylation with Trypanosoma cruzi infection triggering mainly JNK and ERK phosphorylation. Cruzipain also favors the survival in macrophages by changing the iNOS/arginase balance 3.4.22.56 caspase-3 molecular biology engineering of CHO cells for more robust cell lines includes reduction of apoptotic capase-3, overview 3.4.22.59 caspase-6 molecular biology heat sensitive expression of caspase-6 in the embryonic heart is of interest since cardiac malformations are an emergent problem in salmon aquaculture 3.4.22.59 caspase-6 molecular biology it is shown that caspase-6 is the major caspase responsible for the death of cells infected with Adeno-associated virus (AAV) infection 3.4.22.59 caspase-6 molecular biology it is shown that trifolin acetate-induced cell death in human leukemia cells is dependent on caspase-6 3.4.22.59 caspase-6 molecular biology the results show that caspase-1 is an upstream positive regulator of caspase-6-mediated cell death in primary human neurons 3.4.22.60 caspase-7 molecular biology engineering of CHO cells for more robust cell lines includes reduction of apoptotic capase-7, overview 3.4.22.68 Ulp1 peptidase molecular biology modification of recombinant proteins by SUMOylation often dramatically increases solubility and stability during expression of the fusion proteins in bacteria relative to unfused proteins. After expressing a protein as a fusion to SUMO, it is often desirable to cleave the SUMO off of the fusion protein using a SUMO-specific protease such as Ulp1. To facilitate such processing, a dual expression vector is constructed encoding two fusion proteins: one consisting of SUMO fused to Ulp1 and a second consisting of SUMO fused to a His-tagged protein of interest. The SUMO-Ulp1 cleaves both itself and the other SUMO fusion protein in the bacterial cells prior to lysis, and the proteins retain solubility after cleavage, method evaluation, overview 3.4.22.68 Ulp1 peptidase molecular biology usage of the targeting and small ubiquitin-like modifier, SUMO, binding properties of Ulp1(3)(C580S) to purify Smt3-modified proteins from cell extracts 3.4.22.70 sortase A molecular biology a general strategy for the site-specific modification of cell surface proteins with synthetic molecules by using sortase, a transpeptidase from Staphylococcus aureus. The short peptide tag LPETGG is genetically introduced to the C terminus of the target protein, expressed on the cell surface. Subsequent addition of sortase and an N-terminal triglycine-containing probe results in the site-specific labeling of the tagged protein. C-terminal-specific labeling of osteoclast differentiation factor with a biotin- or fluorophore-containing short peptide on the living cell surface. The labeling reaction occurrs efficiently in serum-containing medium, as well as serum-free medium or PBS. The labeled products are detected after incubation for 5 min. In addition, site-specific protein–protein conjugation is successfully demonstrated on a living cell surface by the Sortase-catalyzed reaction. This strategy provides a powerful tool for cell biology and cell surface engineering 3.4.22.70 sortase A molecular biology method for immobilizing ligand proteins onto Biacore sensor chips using the transpeptidase activity of Staphylococcus aureus sortase A. This method provides a robust and gentle approach for the site-directed, covalent coupling of proteins to biosensor chips. The high specificity of the sortase allows immobilization of proteins from less than pure protein samples allowing short cuts in protein purification protocols 3.4.23.B4 Feline immunodeficiency virus protease molecular biology the FIV-PR mutant is a mutational model system to study the molecular basis of substrate-inhibitor specificity for lentivirus proteases, especially HIV-1 protease 3.4.23.5 cathepsin D molecular biology insights on the amino acid region involved in the terminal processing of human cathepsin D and on the function of the processing beta-hairpin loop are provided 3.4.23.21 Rhizopuspepsin molecular biology rhizopuspepsin is a good model enzyme to investigate acid proteinases 3.4.23.34 cathepsin E molecular biology CatE is a potential cancer biomarker 3.4.23.39 plasmepsin II molecular biology plasmepsin 2 but not plasmepsin 4 is a pential tool for hydrogen/deuterium exchange coupled to mass spectroscopy (DXMS) studies 3.4.23.47 HIV-2 retropepsin molecular biology an experimental model system based on the expression of HIV-2 protease in yeast cells is established: HIV-2 protease activity kills the yeast cell, this process can be abolished by inhibiting the viral enzyme activity 3.4.24.11 neprilysin molecular biology the established assay is extremely sensitive to neprilysin, but insensitive, or much less sensitive, to other Abeta-degrading enzymes. As low as 0.1 nM of neprilysin can be detected 3.4.24.12 envelysin molecular biology Ets4 is one of the transcription factors that control HE expression 3.4.24.16 neurolysin molecular biology neurolysin can be used as a molecular tool for analysis of properties of cancer-producing matrix metalloproteinases MMP-2 and MMP-9, quantitative determination of cleavage activity of neurolysin toward MMPspecific fluorescence-quenching peptides, overview 3.4.24.20 peptidyl-Lys metalloendopeptidase molecular biology biological application for enzyme-based proteolytic 18O labeling method characterizing the proteome changes of cytokine/lipolysaccharide-treated versus untreated human retinal pigment epithelium cell line 3.4.24.20 peptidyl-Lys metalloendopeptidase molecular biology proteolytic 18O labeling method employing enzyme for use in comparative proteomics. Enzyme incorporates only a single 18O atom into the carboxyl terminus of each proteolytically generated peptide. Method provides accurate quantification results for isotopically labeled peptides 3.4.24.20 peptidyl-Lys metalloendopeptidase molecular biology a method for detecting protein termini on both the amino and the carboxyl side, regardless of terminal modifications, such as N-acetylation is established. This method requires LC-MS/MS combined with two endopeptidases (lysyl endopeptidase) Lys-C and peptidyl-Lys metalloendopeptidase (Lys-N) 3.4.24.33 peptidyl-Asp metalloendopeptidase molecular biology many eukaryotic proteins are blocked at the alpha-amino group of their N-terminal with various modifications, thereby making it difficult to determine their N-terminal sequence by protein sequencer, development of a method for selectively isolating the blocked N-terminal peptide from the peptide mixture generated by endoproteinase AspN digestion of N-blocked protein by removal of all peptides other than the N-terminal one (non-N-terminal peptides) through their carbonyl group introduced by a chemical transamination reaction 3.4.24.66 choriolysin L molecular biology putative embryonic seCL148 product is most closely related to medaka choriolysin L 3.4.24.69 bontoxilysin molecular biology botulinum neurotoxins BoNT/A-G are widely used as laboratory research tools 3.4.24.79 pappalysin-1 molecular biology heparin administration is associated with a significant increase in PAPP-A levels, presumably because of the detachment of PAPP-A from the vessel wall 3.4.24.79 pappalysin-1 molecular biology mice born with the deletion of the gene for PAPP-A, a model of reduced local IGF activity, live approximately 30% longer than their wild-type littermates. Food intake, and total energy expenditure and resting energy expenditure as measured by calorimetry are not different between PAPP-A knockout and wild-type mice. There is an increase in spontaneous physical activity in PAPP-A knockout mice. Both wild-type and PAPP-A knockout mice exhibit mild insulin resistance with age. Oral glucose tolerance and insulin sensitivity are not significantly different between the two groups of mice, although there appeared to be a decrease in the average size of the pancreatic islets in PAPP-A knockout mice. Thus, neither reduced rate of living nor altered glucose-insulin homeostasis can be considered key determinants of the enhanced longevity of PAPP-A knockout mice 3.4.24.81 ADAM10 endopeptidase molecular biology ADAM10 is regulator of vascular permeability and possesses a function VE-cadherin-dependent endothelial cell functions and leukocyte transendothelial migration 3.4.24.81 ADAM10 endopeptidase molecular biology Kuzbanian, the ADAM10 orthologue in Drosophila melanogaster plays an important role in axon guidance by building a complex with ephrinA2, which is cleaved off from the membrane in a moment of EphA3 receptor binding 3.4.24.81 ADAM10 endopeptidase molecular biology tetraspanins regulate the activity of ADAM10 toward several substrates. It is illustrated how membrane compartmentalization by tetraspanins can control the function of cell surface proteins such as ectoproteases 3.4.24.81 ADAM10 endopeptidase molecular biology there is only a moderate alteration of gene expression in ADAM10 overexpressing mice. Genes coding for pro-inflammatory or pro-apoptotic proteins are not overrepresented among differentially regulated genes. Even a decrease of inflammation markers is observed. This further supports the strategy to treat alzheimer’s disease by increasing the beta-secretase activity 3.4.24.83 anthrax lethal factor endopeptidase molecular biology anthrax lethal toxin treatment of neutrophils disrupts signaling to downstream MAPK targets in response to TLR stimulation. Following anthrax lethal toxin treatment, ERK family and p38 phosphorylation are nearly completely blocked, but signaling to JNK family members persists in vitro and ex vivo. In contrast to previous reports involving human neutrophils, anthrax lethal toxin treatment of murine neutrophils increases their production of superoxide in response to PMA or TLR stimulation in vitro or ex vivo. Although this enhanced superoxide production correlates with effects due to the lethal toxin-induced blockade of ERK signaling, it requires JNK signaling that remains largely intact despite the activity of anthrax lethal toxin 3.4.24.83 anthrax lethal factor endopeptidase molecular biology Bacillus anthracis represses the immune response, in part by altering chromatin accessibility of IL-8 promoter to NFkappaB in epithelial cells. This epigenetic reprogramming, in addition to previously reported effects of lethal toxin, represents an efficient strategy used by Bacillus anthracis for invading the host 3.4.24.83 anthrax lethal factor endopeptidase molecular biology celastrol is identified as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway 3.4.24.83 anthrax lethal factor endopeptidase molecular biology it is shown that treatment of RAW 264.7 murine macrophage cells with anthrax lethaltoxin induces autophagy suggesting a protective role as autophagy inhibition using 3-methyladenine results in an accelerated cell death 3.4.24.83 anthrax lethal factor endopeptidase molecular biology lethal toxin triggers the formation of a membrane-associated inflammasome complex in murine macrophages consisting of caspase-1 and Nalp1b, resulting in cleavage of cytosolic caspase-1 substrates and cell death 3.4.24.83 anthrax lethal factor endopeptidase molecular biology microarray analysis is used to investigate the effects of Bacillus anthracis lethal toxin on human neutrophil-like NB-4 cells to identify markers of intoxication. Genes down-regulated after a 2 h lethal toxin exposure include those encoding chemokines and transcription factors. Significant decreases in the mRNA of interleukin-8, CCL20, CCL3 and CCL4 are observed using real-time PCR. The decreases are more pronounced at 4 and 8 h and are lethal toxin-specific. Decreases in chemokine protein levels are evident after 24 h and are sensitive to low concentrations of lethal toxin. Co-incubation with an anti-lethal factor mAb restores levels of interleukin-8 to 100% and 50%, respectively 3.4.24.83 anthrax lethal factor endopeptidase molecular biology primary keratinocytes are resistant to LeTx cytotoxicity, and MEK cleavage does not correlate with LeTx cytotoxicity. LeTx is considered as an anti-inflammatory agent, however it upregulates RANTES 3.4.24.83 anthrax lethal factor endopeptidase molecular biology proteasome inhibitors block anthrax lethal toxin-mediated caspase-1 activation and can protect against cell death, indicating that the degradation of at least one cellular protein is required for cell death. Proteins can be degraded by the proteasome via the N-end rule. Using amino acid derivatives that act as inhibitors of this pathway, it is shown that the N-end rule is required for anthrax lethal toxin-mediated caspase-1 activation and cell death. The Streptomyces olivoreti peptide bestatin, which inhibits leucine, alanine and arginine aminopeptidases, protects macrophages against anthrax lethal toxin. c-IAP1, a mammalian member of the inhibitor of apoptosis protein (IAP) family is identified, as a novel N-end rule substrate degraded in macrophages treated with anthrax lethal toxin 3.4.24.83 anthrax lethal factor endopeptidase molecular biology protein expression profile of murine macrophages RAW264.7 treated with LeTx is analyzed using two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF MS. Among the differentially expressed spots, cleaved mitogen-activated protein kinase kinase 1 acting as a negative element in the signal transduction pathway, and glucose-6-phosphate dehydrogenase playing a role in the protection of cells from hyperproduction of active oxygen are up-regulated LeTx-treated macrophages 3.4.24.83 anthrax lethal factor endopeptidase molecular biology results suggest that this toxin delivery system is capable of stimulating protective immune responses where effective immunization requires stimulation of both classes of T cells 3.4.24.83 anthrax lethal factor endopeptidase molecular biology the cellular damage inflicted by anthrax lethal toxin depends not only on the innate responses but also on the maturation stage of the cell, which modulates the more general caspase-1-independent responses 3.4.24.83 anthrax lethal factor endopeptidase molecular biology the effects of lethal toxin on the transcriptional regulation of the VCAM1 gene, which contains binding sites in its promoter region for NF-kappaB, IFN regulatory factor-1 (IRF-1), Sp1, GATA-2, and AP-1, in primary human endothelial cells is examined. Lethal toxin enhances cytokine-induced activation of NF-kappaB and IRF-1 which are key factors in the lethal toxin-mediated enhancement of TNF-induced VCAM-1 expression. Altering the activity of key transcription factors involved in host response to infection may be a critical mechanism by which lethal toxin contributes to anthrax pathogenesis 3.4.24.83 anthrax lethal factor endopeptidase molecular biology the in vitro effects of thermal stress on the killing of murine macrophages by anthrax lethal toxin are investigated. Heat shock rapidly halts anthrax lethal toxin-induced cell death without any impact on toxin uptake or mitogen-activated protein kinases cleavage, by a mechanism independent of novel protein synthesis, p38 activation, HSP90 activity or proteasome inhibition. Rather, heat shock prevents the activation of procaspase-1 in anthrax lethal toxin -treated cells, apparently by the sequestration of pro-caspase-1 in a large, inhibitory complex. Heat-shocked cell lysates strongly inhibit the active caspase-1 heterotetramer in vitro, independent of a specific inflammasome platform. Results suggest the presence of a cellular, heat shock-inducible, caspase-1 inhibiting factor 3.4.24.83 anthrax lethal factor endopeptidase molecular biology toxin effects of lethal toxin and edema toxin of Bacillus anthracis in bone marrow dendritic cells stimulated with either LPS or Legionella pneumophila are analysed. Lethal toxin, not ET, is more toxic for cells from BALB/c mice than from C57BL/6 as measured by 7-AAD uptake. Results support the conclusion that lethal toxin and edema toxin are not uniformly suppressive of dendritic cell function but rather modulate function up or down depending on variables such as the function tested, the microbial stimulus used, and the genetic variation in innate immune response mechanisms in the host cell 3.4.24.86 ADAM 17 endopeptidase molecular biology results indicate that oxygen regulates the expression of TACE and TACE may be important for placental development during human pregnancy 3.4.24.86 ADAM 17 endopeptidase molecular biology TACE may be involved in liver regeneration by pathway mediated with transforming growth factor-alpha-epidermal growth factor recptor in the cell-cycle progressive phase in vivo. TACE production and effect by paracrine may be a pathway of involvement in liver regeneration for the activated CD3+ T lymphocytes 3.4.24.86 ADAM 17 endopeptidase molecular biology TACE/ADAM17-like proteases might play a role in synaptic formation to generate specific neuronal connections by processing the excess amount of RA175/SynCAM1, a member of the immunoglobulin family 4, located in the non-synaptic region 3.5.1.88 peptide deformylase molecular biology the combination of plant peptide deformylase and peptide deformylase inhibitors may represent a native gene selectable marker system for chloroplast and nuclear transformation vectors 3.5.4.1 cytosine deaminase molecular biology structure-based, computation-guided predictive method for reversibly controlling enzyme activity using covalently attached photo-responsive azobenzene groups. Application on yeast cytosine deaminase obtains an about 3fold change in enzyme activity by the photo-controlled modulation of the enzyme's active site lid structure, while fully maintaining thermostability. Multiple cycles of switching, controllable in real time, are possible 3.5.4.16 GTP cyclohydrolase I molecular biology mutations in Punch can act as genetic enhancers of Dube3a over-expression phenotypes 3.5.4.16 GTP cyclohydrolase I molecular biology mutations in Punch can act as genetic enhancers of Dube3a overexpression phenotypes 3.5.4.23 blasticidin-S deaminase molecular biology new selection marker for transformation of Arabidopsis thaliana and Nicotiana tabacum 3.5.4.23 blasticidin-S deaminase molecular biology BSD gene will be useful as a new dominant selectable marker for eukaryotes, first sucessful transformation with a drug resistance gene originating from a eukaryote by selecting for detoxification of the drug 3.5.4.23 blasticidin-S deaminase molecular biology generation of bicistronic expression vector mediating BSD resistance, blastidicin S selection of cells 3.5.4.38 single-stranded DNA cytosine deaminase molecular biology a target-AID base editor, designed to recruit cytidine deaminase (CDA) to the target DNA locus via the CRISPR/Cas9 system, can directly induce C to T mutation without double-strand breaks and donor DNA. This system is adopted in Yarrowia lipolytica for multiplex gene disruption. Target-specific gRNA(s) and a fusion protein consisting of a nickase Cas9, CDA1, and uracil DNA glycosylase inhibitor are expressed from a single plasmid to disrupt target genes by introducing a stop codon via C to T mutation within the mutational window. Using this Target-AID system, single gene disruption and simultaneous double gene disruption are achieved with the efficiencies up to 94% and 31%, respectively 3.5.4.42 N-isopropylammelide isopropylaminohydrolase molecular biology hybridization studies localize atzC together with TrzN and atzB to a 380-kb plasmid in Arthrobacter aurescens strain TC1 3.5.99.5 2-aminomuconate deaminase molecular biology nbzE, involved in the ring cleavage pathway of 2-aminophenol, is localized on the 6.6 kb SnaBI-SmaI fragment of the plasmid pNB1 and clusters in the order nbzC-nbzD-nbzE as an operon 3.6.1.1 inorganic diphosphatase molecular biology cycle sequencing methods 3.6.4.B7 RadA recombinase molecular biology genotyping of deep sea isolates and cutured deep-sea hydrothermal vent euryarchaeota 2 isolates using the enzyme sequence 3.7.1.4 phloretin hydrolase molecular biology 98% similarity of the rabbit LPH precursor to PNGH sequence, LPH and PNGH enzymes have the same genomic origin, but differ in transcriptional and, possibly, post-translational processing 3.7.1.4 phloretin hydrolase molecular biology cell specificty of LPH gene expression depends upon both positive and negative interactions among elements in the first 2kb of the LPH 5'-flanking region, generally positive activity between -74 and -37 bp, a cell-specific negative region between -210 and -95 bp, and additional elements further toward the 5' terminus that confer a highly cell-specific response in reporter activity, potential binding sites for various intestinal transcription factors, binding of HNF3beta at three sites is relevant to LPH expression 3.7.1.4 phloretin hydrolase molecular biology LPH and PNGH enzymes have the same genomic origin, but differ in transcriptional and, possibly, post-translational processing 3.7.1.7 beta-diketone hydrolase molecular biology exhibits 63% identity with OPH of Pseudomonas sp. strain VM15C and 29-32% identity with the polyhydroxybutyrate depolymerases from Mesorhizobium loti, Rhizobium sp. and Sinorhizobium meliloti 3.7.1.8 2,6-dioxo-6-phenylhexa-3-enoate hydrolase molecular biology open reading frame corresponding to the pcbD gene consists of 855 base pairs with an ATG initiation codon and a TGA termination codon, able to encode a polypeptide with a molecular weight of 31732 containing 284 amino acid residues, deduced amino acid sequence has 62% identity with those of the HOPDA hydrolases of Pseudomonas putida KF715, P. pseudoalcaligenes KF707, and Burkholderia cepacia LB400, and also significant homology with those of other hydrolytic enzymes including esterase, transferase, and peptidase 3.7.1.8 2,6-dioxo-6-phenylhexa-3-enoate hydrolase molecular biology the deduced amino acid sequence of CarC shows 30.3, 31.3, and 31.8% identity with meta-cleavage compound hydrolases TodF, XylF, and DmpD, respectively, from other Pseudomonas 3.7.1.8 2,6-dioxo-6-phenylhexa-3-enoate hydrolase molecular biology upstream of bphC are five ORFs, including bphD, exhibiting low homology with, and a different gene order from, previously characterized bph genes 3.7.1.9 2-hydroxymuconate-6-semialdehyde hydrolase molecular biology orf234, encoding an alpha/beta hydrolase, which is distantly related to the meta-fission product hydrolases such as XylF, PhnD, and CumD 3.7.1.9 2-hydroxymuconate-6-semialdehyde hydrolase molecular biology the Pseudomonas strutzeri OX1 genes coding for toluene and o-xylene catabolism are organized into at least two operons, the one coding for the phenol catabolism displays a gene order similar to that of the Pseudomonas sp. strain CF600dmp operon, includes HMSH, and is coregulated by the tou operon activator TouR 3.13.2.1 adenosylhomocysteinase molecular biology a coupled fluorescent assay for histone methyltransferase utilizes S-adenosylhomocysteine hydrolase to hydrolyze the methyltransfer product S-adenosylhomocysteine to homocysteine and adenosine. The homocysteine concentration is then determined through conjugation of its free sulfhydryl moiety to a thiol-sensitive fluorophore. the assay allows rapid and facile determination of histine methyltransferase kinetics and can be adapted to measure the enzymatic activity of a wide variety of S-adenosylmethionine-dependent methyltransferases 3.13.2.1 adenosylhomocysteinase molecular biology AdoHcyase overexpression results in elevated adenosine levels and decreased cell viability. Furthermore, AdoHcyase overexpressing cells show different features typical for apoptosis (cell detachment, caspase-like activity, DNA fragmentation), suggesting that cell death is due to apoptosis 3.13.2.1 adenosylhomocysteinase molecular biology Streptococcal pyrogenic exotoxin B cleaves human S-adenosylhomocysteine hydrolase and induces hypermethioninemia 4.1.1.23 orotidine-5'-phosphate decarboxylase molecular biology usage of the gene as a selection marker 4.1.1.23 orotidine-5'-phosphate decarboxylase molecular biology the orotidine 5'-phosphate decarboxylase encoding gene, pm-ura3, from Pestalotiopsis microspora strain NK17 is utilized as a marker in the construction of a reusable system for gene scarless deletion, restoration, and tagging. No heterogeneous markers are needed, and the accumulation of foreign DNA sequences such as HisG, FRP, and LoxP is avoided 4.1.1.39 ribulose-bisphosphate carboxylase molecular biology a continuous assay for Rubisco activity in crude cell extracts using the Mn2+ chemiluminescence of Rubisco oxygenase is described 4.1.1.39 ribulose-bisphosphate carboxylase molecular biology a membrane inlet mass spectrometer method is developed that simultaneously determines the rate of Rubisco carboxylation (vc) and oxygenation (vo) and the CO2 and O2 concentrations 4.1.1.39 ribulose-bisphosphate carboxylase molecular biology a membrane inlet mass spectrometer method is developped that simultaneously determines the rate of Rubisco carboxylation (vc) and oxygenation (vo) and the CO2 and O2 concentrations 4.1.1.39 ribulose-bisphosphate carboxylase molecular biology proteome analysis of peanut leaf is conducted using two-dimensional gel electrophoresis in combination with sequence identification using MALDI/TOF. A master leaf polypeptide profile is generated based on the consistently expressed protein pattern. Proteins present in 205 spots are identified, including RuBisCO 4.1.2.13 fructose-bisphosphate aldolase molecular biology the enzyme gene controls the flux of key intermediates for oil biosynthesis 4.1.3.1 isocitrate lyase molecular biology cell growth in presence of high salt concentrations (1 M NaCl or KCl) leads to increase in enzyme activity consisting with higher levels of succinate and decreased levels of isocitrate 4.1.3.1 isocitrate lyase molecular biology glyoxylate shunt crucial for NADH/NADPH conversion cycle 4.1.3.27 anthranilate synthase molecular biology use of the feedback-insensitive alpha-subunit OASA1D (N323D) of anthranilate synthase as a selectable marker for transformation of rice and potato, the selection system will prove applicable to a wide range of plant species and culture procedures 4.2.1.11 phosphopyruvate hydratase molecular biology alpha-enolase doubles as a surface-displayed plasminogen-binder supporting virulence 4.2.1.11 phosphopyruvate hydratase molecular biology influence of enolase on membrane fusion of vacuoles and protein trafficking analyzed 4.2.1.11 phosphopyruvate hydratase molecular biology studies on mitochondrial import machinery of Saccharomyces cerevisiae 4.2.1.104 cyanase molecular biology analyses of conditions to metabolize exogenously supplied cyanate 4.2.1.104 cyanase molecular biology analyses of conditions to metabolize exogenously supplied cyanate, depending on proteins of the Cyn-ABDS operon, light, and on activity of the CO2-concentrating mechanism (CCM), low internal pools of HCO3- and CO2 result in an insufficient supply of bicarbonate 4.2.1.104 cyanase molecular biology analyses of conditions to metabolize exogenously supplied cyanate, depending on proteins of the CynABDS operon, light, and on activity of the CO2-concentrating mechanism (CCM), inactivation of the cynS gene leads to inability of decomposition of external cyanate 4.2.1.104 cyanase molecular biology analyses of conditions to metabolize exogenously supplied cyanate, depending on proteins of the CynABDS operon, light, and on activity of the CO2-concentrating mechanism (CCM), mutagensis of a periplasmatic binding protein of a multicomponent ABC-transporter (CynA), leads to inability of decomposition of external cyanate due to impaired cyanate uptake, cyanase function is not affected 4.2.1.104 cyanase molecular biology analyses of conditions to metabolize exogenously supplied cyanate, depending on proteins of the operon CynABDS, light and internal pools of HCO3- and CO2 4.2.2.2 pectate lyase molecular biology pectate lyase plays an important degradative role in the primary wall and middle lamella in ripening strawberry fruit, and should be included in synergistic models of cell wall disassembly 4.2.2.2 pectate lyase molecular biology pH-regulated response is only part of a multi-factor regulation of PELB. Sugars are also needed to promote the transition from quiescent to active necrotrophic development by the pathogen 4.2.2.2 pectate lyase molecular biology the increase in expression of both pectate lyase correlates well with the decrease in firmness observed in the fruit 4.2.2.2 pectate lyase molecular biology virulence factors, including pectate lyase (Pel),exoprotease, tabtoxin, and syringomycin production, are found to be regulated by GacS/GacA homologues in phytopathogens 4.2.2.3 mannuronate-specific alginate lyase molecular biology this is the first report that yeast cells displaying alginate lyase are used to produce different lengths of oligosaccharides from alginate 4.2.2.20 chondroitin-sulfate-ABC endolyase molecular biology glycosaminoglycans are extracted from cooked haddock muscle. Reverse phase chromatography and digestion with chondroitinase ABC (Chase) is used. FeCl3 is mixed with the purified glycosaminoglycans, and Fe uptake is measured by ferritin formation using an in vitro digestion/Caco-2 cell model. The identificative analyses suggest that chondroitin/dermatan sulfate-related structures promote Fe uptake by Caco-2 cells 4.3.1.18 D-Serine ammonia-lyase molecular biology the dsdA gene is used as a selectable marker for transformation of Arabidopsis 4.3.1.19 threonine ammonia-lyase molecular biology in contrast to the wild-type, all four transgenic TD lines are able to tolerate high concentrations of L-O-methylthreonine. This illustrates the potential use of these mutant omr genes as dominant selectable markers in plant transformation 4.4.1.17 Holocytochrome-c synthase molecular biology system III, cytochrome c heme lyase, is an enzyme found in the mitochondria of many eukaryotes, which is used for heterologous expression of mitochondrial holocytochromes c 4.4.1.20 leukotriene-C4 synthase molecular biology the third hydrophobic region, containing a putative transmembrane helix, acts as a nuclear envelope localization signal and is involved in the homooligomerization of LTC4S 4.4.1.20 leukotriene-C4 synthase molecular biology TNF-alpha exposure downregulates the LTC4 synthase gene expression in monocytes/macrophages via a transcriptional mechanism 4.4.1.20 leukotriene-C4 synthase molecular biology transient transfection into human monocytic leukemia (THP-1), rat basophilic leukemia (RBL-1), and human embryonic kidney (HEK293/T) epithelial cells show that eGFP was expressed by cells which express leukotriene C4 synthase (RBL-1 and THP-1) but not by the leukotriene C4 synthase negative HEK293/T cells 4.6.1.1 adenylate cyclase molecular biology the enzyme is a favorable optogenetic tools for non-invasive, cell-selective, and spatio-temporally precise modulation of cAMP/cGMP with light. The rhodopsin domain from Catenaria is more photostable than that from Blastocladiella, and the signaling state persists longer, both of which are highly desirable traits for optogenetic applications 4.6.1.2 guanylate cyclase molecular biology the enzyme is a favorable optogenetic tool for non-invasive, cell-selective, and spatio-temporally precise modulation of cAMP/cGMP with light. The rhodopsin domain from Catenaria is more photostable than that from Blastocladiella, and the signaling state persists longer, both of which are highly desirable traits for optogenetic applications 4.6.1.2 guanylate cyclase molecular biology the enzyme is a favorable optogenetic tools for non-invasive, cell-selective, and spatio-temporally precise modulation of cAMP/cGMP with light. The rhodopsin domain from Catenaria is more photostable than that from Blastocladiella, and the signaling state persists longer, both of which are highly desirable traits for optogenetic applications 4.6.1.13 phosphatidylinositol diacylglycerol-lyase molecular biology enzyme is used as a tool in the studies of GPI-anchored proteins 4.6.1.16 tRNA-intron lyase molecular biology controlled RNA splicing in mammalian cells. mRNA modification technology that makes use of tRNA splicing endonuclease and its natural substrate, the bulge-helix-bulge structure. These components can perform both cis- and trans-splicing in cellular and animal models and may provide a convenient way to modulate gene expression using components independent of cellular regulatory networks. To use the Methanocaldococcus jannaschii enzyme in stable expression mammalian systems, variants are developed which are characterized by high efficiency and sustainable in vivo activity. The variants are created by the introduction of proper localization signals followed by mutagenesis and direct selection in mammalian cells. The best endonuclease variant shows 40fold higher activity compared to the parental enzyme and stable processing of 30% of the target mRNA. These variants show complete compatibility with long-term expression in mammalian cells, suggesting that they may be usefully applied in functional genomics and genetically modified animal models 5.1.1.5 lysine racemase molecular biology the enzyme is valuable to serve as a novel non-antibiotic selectable marker in the generation of transgenic plants 5.1.1.8 4-hydroxyproline epimerase molecular biology PrpA can be used as a protein adjuvant in a live Salmonella delivery system, in order to increase humoral responses effectively without major interference on the cell mediated immunity 5.4.2.2 phosphoglucomutase (alpha-D-glucose-1,6-bisphosphate-dependent) molecular biology phosphoglucomutase is a very suitable marker for analysis of the inter-breed and intra-breed polymorphism for the mulberry silkworm, and for determining the level of genetic variability 5.4.99.B25 tRNA pseudouridine54 synthase molecular biology using the COG1901 recombinant enzyme from Methanocaldococcus jannaschii (Mj1640), purified enzyme Pus10 from Methanocaldococcus jannaschii and full-size tRNA transcripts or T(PSI)-arm (17-mer) fragments as substrates, the sequential pathway of m(1)PSI54 formation in Archaea is reconstituted 5.6.1.7 chaperonin ATPase molecular biology CPN10 and CPN60.2 are suitable markers for the mitochondria of Leishmania spp. 5.6.1.7 chaperonin ATPase molecular biology the development of a spectroscopic actin folding assay that allows actin folding by CCT to be monitored in real time through labeling with the environmentally sensitive dye acrylodan. This approach allows greater sensitivity in the measurement of actin folding kinetics than gel electrophoresis-based assays, and is used to observe the effects of ATP concentration and temperature on the rate of actin folding 6.1.1.5 isoleucine-tRNA ligase molecular biology enzyme is a target for receptor-guided inhibitor design 6.1.1.7 alanine-tRNA ligase molecular biology a combined action of ligases MurM and MurN is required in order to rationalise the high level of dipeptide cross-links in penicillin-resistant Streptococcus pneumoniae, with ligase MurM showing the major difference between penicillin-resistant and penicillin-sensitive strains 6.1.1.7 alanine-tRNA ligase molecular biology the unique widespread distribution of the free-standing editing domain homolog AlaXp is most probably due to singular difficulties, for translation, poised by alanine 6.1.1.11 serine-tRNA ligase molecular biology the role of VlmL in valanimycin biosynthesis is to produce L-seryl-tRNA for the valanimycin biosynthetic pathway 6.1.1.14 glycine-tRNA ligase molecular biology translation of mRNA for yeast glycyl-tRNA synthetase is alternatively initiated from UUG and a downstream AUG initiation codon. Unlike an AUG initiation codon, efficiency of this non-AUG initiation codon is significantly affected by its sequence context, in particular the nucleotides at positions -3 to -1 relative to the initiation codon. A/A/R (R: A or G) and C/G/C appear to be the most and least favorable sequences at these positions, respectively. Mutation of the native context sequence -3 to -1 from AAA to CGC reduce translation initiation from the UUG codon up to 32fold and resulted in loss of mitochondrial respiration 6.1.1.16 cysteine-tRNA ligase molecular biology Mycoplasmas hyopneumoniae are collected from bronchial alveolar lavage samples of infected pigs 28 days postinfection and compared to broth-grown cells using microarrays. During lung infection, the analysis indicates that 79 genes are differentially expressed. Of the down-regulated genes, 28 of 46 (61%) lacked an assigned function, in comparison to 21 of 33 (63%) of up-regulated genes. Cysteinyl-tRNA synthetase is down-regulated in vivo 6.1.1.26 pyrrolysine-tRNAPyl ligase molecular biology the pyrrolysyl-tRNA synthetase/tRNAPyl suppression system can be used for the in vitro synthesis of peptides with nonnatural backbones 6.1.1.26 pyrrolysine-tRNAPyl ligase molecular biology PylRS as aminoacyl-tRNA synthetase allows the Pyl incorporation machinery to be easily engineered for the genetic incorporation of more than 100 non-canonical amino acids (NCAAs) or alpha-hydroxy acids into proteins at amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and four-base AGGA codons to code NCAAs 6.2.1.5 succinate-CoA ligase (ADP-forming) molecular biology the maltose-binding protein-tagged beta-subunit of succinyl-CoA synthetase, SUCLA2, bound to an amylose resin, is used as an affinity ligand to bind FPLC-purified wild-type and some mutant erythroid-specific aminolevulinic acid synthases, ALAS2 6.3.1.20 lipoate-protein ligase molecular biology dependency of pathogen Listeria monocytogenes on LplA1 dictated by availability of host lipoyl substrates as alternative lipoate source 6.3.4.5 argininosuccinate synthase molecular biology report in which the function of a Porphyra yezoensis gene has been directly demonstrated by the rescue of a Saccharomyces cerevisiae mutant. This technique may provide new opportunities for further investigations into the functions of various genes in Porphyra yezoensis and other macroalgal species 6.5.1.1 DNA ligase (ATP) molecular biology LihTh1519 may be used for basic and applied researches in molecular biology and genetic engineering 6.5.1.1 DNA ligase (ATP) molecular biology inclusion of the C-terminally 6His-tagged PabDBD in the reaction mixture during long-range polymerase chain reaction (PCR) increases the efficacy of amplification and eliminates the inhibitory effect of heparin 6.5.1.3 RNA ligase (ATP) molecular biology RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) 6.5.1.3 RNA ligase (ATP) molecular biology the properties of TS2126 RNA ligase 1 makes it very attractive for processes like adaptor ligation, and single-stranded solid phase gene synthesis 7.1.2.2 H+-transporting two-sector ATPase molecular biology F1FO-ATP synthase is a Na+-translocating ATPase used to generate an electrochemical gradient of Na+ that can drive other membrane-bound bioenergetic processes 7.6.2.1 P-type phospholipid transporter molecular biology phosphatidylserine exposure by Tat-1 is an early in vivo marker of apoptosis in Caenorhabditis elegans 7.6.2.9 ABC-type quaternary amine transporter molecular biology opuC gene is transcribed exclusively from a sigmaB-dependent promoter, transcripts originating from the sigmaB-dependent opuC accumulate substantially after osmotic upshift but are minimal after temperature upshift or ethanol stress