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Intra and extracellular nuclease production by Aspergillus niger and Aspergillus nidulans
Ferreira, Adlane V. B.;
Revista de Microbiologia , 1998, DOI: 10.1590/S0001-37141998000400009
Abstract: intra and extracellular nuclease production by strains of aspergillus niger and aspergillus nidulans was estimated using a modified dnase test agar and cell-free extract assays. differences in the production of nucleases by a. niger and a. nidulans were observed. these observations suggest that the dnase test agar can be helpful for a quick screening for some types of nucleases in filamentous fungi. the assays using cell-free extracts can also be useful for initial characterization of other types of nucleases.
Intra and extracellular nuclease production by Aspergillus niger and Aspergillus nidulans  [cached]
Ferreira Adlane V. B.
Revista de Microbiologia , 1998,
Abstract: Intra and extracellular nuclease production by strains of Aspergillus niger and Aspergillus nidulans was estimated using a modified DNAse test agar and cell-free extract assays. Differences in the production of nucleases by A. niger and A. nidulans were observed. These observations suggest that the DNAse test agar can be helpful for a quick screening for some types of nucleases in filamentous fungi. The assays using cell-free extracts can also be useful for initial characterization of other types of nucleases.
Sequence Specificity of BAL 31 Nuclease for ssDNA Revealed by Synthetic Oligomer Substrates Containing Homopolymeric Guanine Tracts  [PDF]
April Marrone, Jack Ballantyne
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0003595
Abstract: Background The extracellular nuclease from Alteromonas espejiana, BAL 31 catalyzes the degradation of single-stranded and linear duplex DNA to 5′-mononucleotides, cleaves negatively supercoiled DNA to the linear duplex form, and cleaves duplex DNA in response to the presence of apurinic sites. Principal Findings In this work we demonstrate that BAL 31 activity is affected by the presence of guanine in single-stranded DNA oligomers. Specifically, nuclease activity is shown to be affected by guanine's presence in minimal homopolymeric tracts in the middle of short oligomer substrates and also by its presence at the 3′ end of ten and twenty base oligomers. G?C rich regions in dsDNA are known to cause a decrease in the enzyme's nuclease activity which has been attributed to the increased thermal stability of these regions, thus making it more difficult to unwind the strands required for enzyme access. Our results indicate that an additional phenomenon could be wholly or partly responsible for the loss of activity in these G?C rich regions. Thus the presence of a guanine tract per se impairs the enzyme's functionality, possibly due to the tract's bulky nature and preventing efficient progression through the active site. Conclusions This study has revealed that the general purpose BAL 31 nuclease commonly used in molecular genetics exhibits a hithertofore non-characterized degree of substrate specificity with respect to single-stranded DNA (ssDNA) oligomers. Specifically, BAL 31 nuclease activity was found to be affected by the presence of guanine in ssDNA oligomers.
Mung Bean Nuclease Treatment Increases Capture Specificity of Microdroplet-PCR Based Targeted DNA Enrichment  [PDF]
Zhenming Yu, Kajia Cao, Tanya Tischler, Catherine A. Stolle, Avni B. Santani
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0103491
Abstract: Targeted DNA enrichment coupled with next generation sequencing has been increasingly used for interrogation of select sub-genomic regions at high depth of coverage in a cost effective manner. Specificity measured by on-target efficiency is a key performance metric for target enrichment. Non-specific capture leads to off-target reads, resulting in waste of sequencing throughput on irrelevant regions. Microdroplet-PCR allows simultaneous amplification of up to thousands of regions in the genome and is among the most commonly used strategies for target enrichment. Here we show that carryover of single-stranded template genomic DNA from microdroplet-PCR constitutes a major contributing factor for off-target reads in the resultant libraries. Moreover, treatment of microdroplet-PCR enrichment products with a nuclease specific to single-stranded DNA alleviates off-target load and improves enrichment specificity. We propose that nuclease treatment of enrichment products should be incorporated in the workflow of targeted sequencing using microdroplet-PCR for target capture. These findings may have a broad impact on other PCR based applications for which removal of template DNA is beneficial.
Staphylococcus aureus Nuc2 Is a Functional, Surface-Attached Extracellular Nuclease  [PDF]
Megan R. Kiedrowski, Heidi A. Crosby, Frank J. Hernandez, Cheryl L. Malone, James O. McNamara, Alexander R. Horswill
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0095574
Abstract: Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc) enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the development of an infection imaging approach based on Nuc-activatable probes. Interestingly, S. aureus strains encode a second nuclease (Nuc2) that has received limited attention. With the growing interest in bacterial nucleases, we sought to characterize Nuc2 in more detail through localization, expression, and biochemical studies. Fluorescence microscopy and alkaline phosphatase localization approaches using Nuc2-GFP and Nuc2-PhoA fusions, respectively, demonstrated that Nuc2 is membrane bound with the C-terminus facing the extracellular environment, indicating it is a signal-anchored Type II membrane protein. Nuc2 enzyme activity was detectable on the S. aureus cell surface using a fluorescence resonance energy transfer (FRET) assay, and in time courses, both nuc2 transcription and enzyme activity peaked in early logarithmic growth and declined in stationary phase. Using a mouse model of S. aureus pyomyositis, Nuc2 activity was detected with activatable probes in vivo in nuc mutant strains, demonstrating that Nuc2 is produced during infections. To assess Nuc2 biochemical properties, the protein was purified and found to cleave both single- and double-stranded DNA, and it exhibited thermostability and calcium dependence, paralleling the properties of Nuc. Purified Nuc2 prevented biofilm formation in vitro and modestly decreased biomass in dispersal experiments. Altogether, our findings confirm that S. aureus encodes a second, surface-attached and functional DNase that is expressed during infections and displays similar biochemical properties to the secreted Nuc enzyme.
Antifungal Potential of Extracellular Metabolites Produced by Streptomyces hygroscopicus against Phytopathogenic Fungi
Benjaphorn Prapagdee, Chutima Kuekulvong, Skorn Mongkolsuk
International Journal of Biological Sciences , 2008,
Abstract: Indigenous actinomycetes isolated from rhizosphere soils were assessed for in vitro antagonism against Colletotrichum gloeosporioides and Sclerotium rolfsii. A potent antagonist against both plant pathogenic fungi, designated SRA14, was selected and identified as Streptomyces hygroscopicus. The strain SRA14 highly produced extracellular chitinase and β-1,3-glucanase during the exponential and late exponential phases, respectively. Culture filtrates collected from the exponential and stationary phases inhibited the growth of both the fungi tested, indicating that growth suppression was due to extracellular antifungal metabolites present in culture filtrates. The percentage of growth inhibition by the stationary culture filtrate was significantly higher than that of exponential culture filtrate. Morphological changes such as hyphal swelling and abnormal shapes were observed in fungi grown on potato dextrose agar that contained the culture filtrates. However, the antifungal activity of exponential culture filtrates against both the experimental fungi was significantly reduced after boiling or treatment with proteinase K. There was no significant decrease in the percentage of fungal growth inhibition by the stationary culture filtrate that was treated as above. These data indicated that the antifungal potential of the exponential culture filtrate was mainly due to the presence of extracellular chitinase enzyme, whereas the antifungal activity of the stationary culture filtrate involved the action of unknown thermostable antifungal compound(s).
Probabilistic Approach to Predicting Substrate Specificity of Methyltransferases  [PDF]
Teresa Szczepińska,Jan Kutner,Micha? Kopczyński,Krzysztof Paw?owski,Andrzej Dziembowski,Andrzej Kudlicki,Krzysztof Ginalski ,Maga Rowicka
PLOS Computational Biology , 2014, DOI: doi/10.1371/journal.pcbi.1003514
Abstract: We present a general probabilistic framework for predicting the substrate specificity of enzymes. We designed this approach to be easily applicable to different organisms and enzymes. Therefore, our predictive models do not rely on species-specific properties and use mostly sequence-derived data. Maximum Likelihood optimization is used to fine-tune model parameters and the Akaike Information Criterion is employed to overcome the issue of correlated variables. As a proof-of-principle, we apply our approach to predicting general substrate specificity of yeast methyltransferases (MTases). As input, we use several physico-chemical and biological properties of MTases: structural fold, isoelectric point, expression pattern and cellular localization. Our method accurately predicts whether a yeast MTase methylates a protein, RNA or another molecule. Among our experimentally tested predictions, 89% were confirmed, including the surprising prediction that YOR021C is the first known MTase with a SPOUT fold that methylates a substrate other than RNA (protein). Our approach not only allows for highly accurate prediction of functional specificity of MTases, but also provides insight into general rules governing MTase substrate specificity.
Modelling substrate specificity and enantioselectivity for lipases and esterases by substrate-imprinted docking
P Benjamin Juhl, Peter Trodler, Sadhna Tyagi, Jürgen Pleiss
BMC Structural Biology , 2009, DOI: 10.1186/1472-6807-9-39
Abstract: Here we present a predictive and robust method to model substrate specificity and enantioselectivity of lipases and esterases that uses reaction intermediates and incorporates protein flexibility. Substrate-imprinted docking starts with covalent docking of reaction intermediates, followed by geometry optimisation of the resulting enzyme-substrate complex. After a second round of docking the same substrate into the geometry-optimised structures, productive poses are identified by geometric filter criteria and ranked by their docking scores. Substrate-imprinted docking was applied in order to model (i) enantioselectivity of Candida antarctica lipase B and a W104A mutant, (ii) enantioselectivity and substrate specificity of Candida rugosa lipase and Burkholderia cepacia lipase, and (iii) substrate specificity of an acetyl- and a butyrylcholine esterase toward the substrates acetyl- and butyrylcholine.The experimentally observed differences in selectivity and specificity of the enzymes were reproduced with an accuracy of 81%. The method was robust toward small differences in initial structures (different crystallisation conditions or a co-crystallised ligand), although large displacements of catalytic residues often resulted in substrate poses that did not pass the geometric filter criteria.The number of protein structures available to researchers has grown exponentially over the last two decades and more than 50 000 experimentally determined structure entries are now held in the Protein Data Bank [1]. Furthermore, comparative structure prediction allows to derive reliable structure models from sequence information [2]. In silico methods are being developed to predict affinity, activity, specificity, and selectivity of newly discovered proteins based on structure information [3]. In drug development, molecular docking is routinely used to identify new lead compounds by virtual screening of libraries of small compounds [4]. Recently, docking methods have also been succes
The Functions of Grainy Head-Like Proteins in Animals and Fungi and the Evolution of Apical Extracellular Barriers  [PDF]
Adam Paré, Myungjin Kim, Michelle T. Juarez, Stuart Brody, William McGinnis
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0036254
Abstract: The Grainy head (GRH) family of transcription factors are crucial for the development and repair of epidermal barriers in all animals in which they have been studied. This is a high-level functional conservation, as the known structural and enzymatic genes regulated by GRH proteins differ between species depending on the type of epidermal barrier being formed. Interestingly, members of the CP2 superfamily of transcription factors, which encompasses the GRH and LSF families in animals, are also found in fungi – organisms that lack epidermal tissues. To shed light on CP2 protein function in fungi, we characterized a Neurospora crassa mutant lacking the CP2 member we refer to as grainy head-like (grhl). We show that Neurospora GRHL has a DNA-binding specificity similar to that of animal GRH proteins and dissimilar to that of animal LSF proteins. Neurospora grhl mutants are defective in conidial-spore dispersal due to an inability to remodel the cell wall, and we show that grhl mutants and the long-known conidial separation-2 (csp-2) mutants are allelic. We then characterized the transcriptomes of both Neurospora grhl mutants and Drosophila grh mutant embryos to look for similarities in the affected genes. Neurospora grhl appears to play a role in the development and remodeling of the cell wall, as well as in the activation of genes involved in defense and virulence. Drosophila GRH is required to activate the expression of many genes involved in cuticular/epidermal-barrier formation. We also present evidence that GRH plays a role in adult antimicrobial defense. These results, along with previous studies of animal GRH proteins, suggest the fascinating possibility that the apical extracellular barriers of some animals and fungi might share an evolutionary connection, and that the formation of physical barriers in the last common ancestor was under the control of a transcriptional code that included GRH-like proteins.
Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1
Qing Song, Xiaobo Zhang
BMC Biotechnology , 2008, DOI: 10.1186/1472-6750-8-43
Abstract: After recombinant expression in Escherichia coli, the purified GBSV1-NSN exhibited non-specific nuclease activity, being able to degrade various nucleic acids, including RNA, single-stranded DNA and double-stranded DNA that was circular or linear. Based on sequence analysis, the nuclease shared no homology with any known nucleases, suggesting that it was a novel nuclease. The characterization of the recombinant GBSV1-NSN showed that its optimal temperature and pH were 60°C and 7.5, respectively. The results indicated that the enzymatic activity was inhibited by enzyme inhibitors or detergents, such as ethylene diamine tetraacetic acid, citrate, dithiothreitol, β-mercaptoethanol, guanidine hydrochloride, urea and SDS. In contrast, the nuclease activity was enhanced by TritonX-100, Tween-20 or chaps to approximately 124.5% – 141.6%. The Km of GBSV1-NSN nuclease was 231, 61 and 92 μM, while its kcat was 1278, 241 and 300 s-1 for the cleavage of dsDNA, ssDNA and RNA, respectively.Our study, therefore, presented a novel thermostable non-specific nuclease from thermophilic bacteriophage and its overexpression and purification for scientific research and applications.Nucleases are defined as a group of enzymes which are capable of hydrolyzing the phosphodiester linkages of nucleic acids. According to the substrates they hydrolyze, nucleases are divided into two groups: sugar specific nucleases (deoxyribonucleases and ribonucleases) and sugar non-specific nucleases [1]. Sugar non-specific nucleases, characterized by their ability to hydrolyze both DNA and RNA without exhibiting pronounced base preferences, have been detected from a wide variety of sources, such as virus, bacteria, fungi and animals [1-4]. Many of them are extracellular, but some have been found in nuclei, vacuoles, mycelia, mitochondria, conidia, microplasmodia and periplasm [5-11]. Sugar non-specific nucleases play very important roles in different aspects of basic genetic mechanisms, including their parti
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