site directed and random insertional mutagenesis in medically important fungi
Báo cáo y học: "Successful tumour necrosis factor (TNF) blocking therapy suppresses oxidative stress and hypoxiainduced mitochondrial mutagenesis in inflammatory arthritis" pot
... temperature for 20 minutes, fixed in acetone for 10 minutes and washed in PBS for minutes Non-specific binding was blocked using 10% casein in PBS for 20 minutes The sections were incubated with primary ... visualise staining, and Mayer’s haematoxylin (BDH Laboratories, Poole, UK) was incubated for 30 seconds as a counterstain prior to mounting in Pertex mounting media Images were captured using Olympus ... significant baseline inverse correlation between tpO2 measurements and 4HNE expression [20] In this study we extend these findings and demonstrate that change in tpO is also significantly and inversely...
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Inducible targeted tagging system for localized insertional mutagenesis in arabidopsis thaliana
... most closely related serine/threonine protein kinases 93 Figure 33: Phylogenetic tree for Serine/Threonine protein kinases 94 Figure 34: GUS staining in L267; an insertion in the sense orientation ... 1.7.1 Random tagging (gene trap) The approach of random tagging incorporates introduction of an autonomous and a non-autonomous element transgenically into plants followed by crossing them to initiate ... of GUS staining for L479; an insertion in the sense orientation in At2g30830 80 Figure 27: A schematic representation of GUS staining in L227; an insertion in the sense orientation in At2g30840...
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Aerobic Uptake of Cholesterol by Ergosterol Auxotrophic Strains in Candidaglabrata & Random and Site-Directed Mutagenesis of ERG25 in Saccharomyces cerevisiae
... Ergosterol Auxotrophic Strains in Candida glabrata & Random and SiteDirected Mutagenesis of ERG25 in Saccharomyces cerevisiae Major Professor: Dr Martin Bard Candida albicans and Candida glabrata are ... strain SCY876 Site- Directed mutagenesis focused on the four histidine clusters, the KKXX C-terminal motif and other conserved amino acids among various plant, animal, and fungal species Random mutagenesis ... Site- directed mutagenesis amino acid change, complementation, and GC results 69 4.3 GC sterol profile values for complementing site- directed mutagenesis strains 70 4.4 Sequence results for random mutagenesis...
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Báo cáo Y học: Potential active-site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site-directed mutagenesis ppt
... (Darmstadt, Germany) Site- directed mutagenesis Site- directed mutagenesis of (His)6PNAE was achieved using the QuikChangeTM in vitro Site- Directed Mutagenesis Kit, according to the manufacturer’s ... of PNAE In order to gain evidence for these suggestions, inhibitor studies, site- directed mutagenesis experiments and modelling of PNAE were performed Inhibitor studies Several known inhibitors ... Site- directed mutagenesis In this study, we have produced wild-type (His)6PNAE and 10 muteins of it Four of six cysteine residues in positions 20, 132, 170 and 257 were changed to alanine and...
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Tài liệu Báo cáo khoa học: nsights into the reaction mechanism of glycosyl hydrolase family 49 Site-directed mutagenesis and substrate preference of isopullulanase doc
... reportedly located in the vicinity of the active site and could form a binding site for a glucosyl unit in subsite )1 (Fig 4B) Together with the observations from the site- directed mutagenesis, Trp402 ... a solution in the Dali result It is likely that the interface between N-terminal and b-helical domains participate in binding of the polysaccharide, pullulan and 396 are positioned in a potential ... participating in subsite )1 The residues that form potential subsites )1 and )2 are reportedly more conserved in GH family 49 than the residues in subsites +1 and +2 [17] Comparison of the active sites...
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Tài liệu Báo cáo khoa học: Identification of critical active-site residues in angiotensin-converting enzyme-2 (ACE2) by site-directed mutagenesis docx
... Chloride binding to ACE2 (yellow) and tACE (white) (A) Binding site of CL1 in ACE2 and tACE; (B) binding site of CL2 in ACE2 and tACE Residue numbering for ACE2 is first The chloride ion is green and ... the binding of chloride to this site might be expected to affect zinc and ligand binding as well as the interactions between subdomain I and II This effect would only be present in ACE as the site ... chloride ion in tACE] Arg514 in ACE2 is displaced relative to Arg522 in ACE and is somewhat closer to the zinc-binding site The CL2 binding site is in close proximity to the catalytic site ˚ (10.4...
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Tài liệu Báo cáo khoa học: Insights into the interaction of human arginase II with substrate and manganese ions by site-directed mutagenesis and kinetic studies Alteration of substrate specificity by replacement of Asn149 with Asp docx
... agmatine Agmatine, which results from decarboxylation of arginine by arginine decarboxylase, is a metabolic intermediate in the biosynthesis of putrescine and higher polyamines and may have important ... residues known to be involved in binding and hydrolysis of the guanidino group of l-arginine by arginase are strictly conserved in the active site of the agmatinases [19] Moreover, modeling studies have ... substrate l-arginine [11,13] Particularly interesting has been a possible role of arginase II in regulating the availability of l-arginine for nitric oxide synthesis in human penile and clitoral...
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Tài liệu Báo cáo Y học: Insights into the reaction mechanism of Escherichia coli agmatinase by site-directed mutagenesis and molecular modelling ppt
... corrected by hand Since the sequences of the two arginases and agmatinase greately differ in the N terminal region, the agmatinase and rat liver arginases were stripped of the first 32 and five amino acids, ... differences between agmatinase and arginase, agmatine and arginine were fixed in essentially the same position in the corresponding active site The same position for the scissile guanidine carbon of the ... differences in this loop area are key factors in determining the difference in substrate specificity between arginase and agmatinase This aspect is presently under investigation in our laboratory In any...
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Báo cáo khoa học: Characterization of HbpR binding by site-directed mutagenesis of its DNA-binding site and by deletion of the effector domain pot
... is important in forming the oligomeric structure and for DNA binding, by cloning an hbpR gene devoid of the A-domain, purifying this protein, and analyzing its DNA-binding characteristics and ... motifs in the binding site for HbpR and to examine the necessity of the sensing A-domain of HbpR for DNA binding The role of individual nucleotides and nucleotide motifs was characterized by site- directed ... protein was correctly expressed in E coli DNA-binding characteristics of the DA-HbpR protein EMSA performed on the wild-type HbpR-binding site and increasing amounts of DA-HbpR or HbpR in fusion...
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Báo cáo khoa học: Residues affecting the chloride regulation and substrate selectivity of the angiotensin-converting enzymes (ACE and ACE2) identified by site-directed mutagenesis pot
... (A) and ACE2 (B) was visualized by immunoblotting using specific human polyclonal antibodies Y224/ 204 Fig Chloride-binding sites of tACE (orange) and ACE2 (red): (A) CL1 binding site; (B) CL2 binding ... 6034 site of sACE, the roles of the equivalent tACE and ACE2 residues have not been investigated previously In this study, candidate residues potentially involved in chloride binding at the CL1 and ... mutagenesis of CL1 and CL2 site residues of tACE and ACE2 The residues surrounding the chloride ion at the CL1 and CL2 sites of tACE and ACE2 are shown in Fig In tACE, the residues that coordinate the...
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Báo cáo khoa học: Investigation of the substrate specificity of a b-glycosidase from Spodoptera frugiperda using site-directed mutagenesis and bioenergetics analysis pdf
... (dotted lines) involving the residue 39 and E451 were represented 4-OH is represented in the equatorial (4e; pointing towards left) and axial (4a; pointing upwards) positions 3-OH is always in the ... residues, E and N, were introduced at position 39, originally occupied by Q Glutamine and asparagine present similar side-chains, containing hydrogen bond donor and acceptor atoms (Nd, Ne and Od, ... Materials and methods All reagents, unless otherwise specified, were purchased from Sigma or Merck Site- directed mutagenesis Site- directed mutagenesis was performed using a plasmid pT7-7 [13] containing...
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Báo cáo khoa học: Probing suggested catalytic domains of glycosyltransferases by site-directed mutagenesis Tobias Hefner and Joachim Stockigt ¨ ppt
... residue in the same position as histidine occupies in inverting transferases (position 360), it would be an exciting challenge to convert an inverting to a retaining enzyme just by such a point mutation ... from cloning Fig Arbutin synthase amino acid sequence showing the NRD1bS (boxed) and NRD1bL (dot boxed) domains, putative DxD motifs (written white on black), and the H–E site in position and position ... stained SDS/PAGE [4] Protein concentration and activity Protein concentrations were measured using the method of Bradford [5] and a standard curve derived from bovine serum albumin For testing...
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Báo cáo Y học: Role of tyrosine 238 in the active site of Rhodotorula gracilis D-amino acid oxidase A site-directed mutagenesis study docx
... side chain of this active site residue in substrate/ligand xation The ligand-binding experiments demonstrate that the overall substrate-binding pocket remains intact, as all mutants bind the ... wild-type and Y238S, the Y238F RgDAAO mutant showed a signicant increase in the intensity of the ễcharge transferế absorbance band at % 600 nm following the binding of anthranilate (Fig 3, inset) and ... inferred for Y223 in binding and xation was suggested [4] This proposal was changed recently following the investigation of the effect of pH on benzoate binding in RgDAAO [12] and the resolution...
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Báo cáo khoa học: Site-directed mutagenesis and footprinting analysis of the interaction of the sunflower KNOX protein HAKN1 with DNA ppt
... single -site mutations on HAKN1 binding to TGACA and variants of this sequence It is logical to assume that changes in amino acids involved in the interaction must in uence binding efficiency In ... analysis of HAKN1 binding to DNA An oligonucleotide containing two HAKN1 binding sites (BS1) in opposite orientations was labeled in the 3¢ end of either strand (HindIII or XbaI sites) and subjected ... oligonucleotide contains two sites in opposite orientation, both strands of the binding site can be observed in a single footprinting assay Analysis of the cleavage patterns indicates that HAKN1...
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Báo cáo khoa học: Defining the QP-site of Escherichia coli fumarate reductase by site-directed mutagenesis, fluorescence quench titrations and EPR spectroscopy doc
... FrdC and FrdD play important roles in defining the QP site In every case where binding is detected, the data can be fitted to an equation (Eqn 1) describing noncooperative binding at a single site ... membraneintrinsic domain of SdhCDAB coordinates a single heme b (b556) that is sandwiched between the SdhC and SdhD subunits [8,9] Quinone binding and reduction is believed to take place in the ... indicates that the mutant is only able to accept a single electron from MQH2, resulting in a bound and stabilized menasemiquinone intermediate, thus explaining the observed binding of HOQNO and...
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Efficient site directed in vitro mutagenesis using ampicillin selection potx
... site in the Testing pSELECT-1 with an Insert In order to test the functioning of pSELECT- with an insert, we first deleted the Eco R1 site in the linker by cutting with this enzyme, filling in ... oligo A single base deletion creating an Eco R1 site and a single base insertion creating a Cla site The insertion and deletion were separated on the mutagenic oligo by bases After single strand DNA ... by propagating the plasmid in E coli NM522 and infecting with M13K07 helper phage In vitro mutagenesis to remove the Hind Im site was performed by hybridizing an oligonucleotide having the sequence...
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Báo cáo khoa học: Site-directed mutagenesis, kinetic and inhibition studies of aspartate ammonia lyase from Bacillus sp. YM55-1 pptx
... with an alanine or serine (T101A and T101S), Asn142 with an alanine or glutamine (N142A and N142Q) and His188 with an alanine, glutamine, lysine or arginine (H188A, H188Q, H188K and H188R) These ... T187S), Asn326 with an alanine or glutamine (N326A and N326Q) and Lys324 with an alanine, serine, valine, histidine, arginine or aspartic acid (K324A, K324S, K324V, K324H, K324R and K324D) It was found ... effects, including the suboptimal positioning of the amino group and the slower release of product In the substrate binding model of AspB, two other residues (Thr101 and Asn142) are implicated in binding...
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Báo cáo khoa học: Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis potx
... Trp244 and Asp245, to coenzyme binding and specificity in P asaccharolyticus GDH Five mutants were created: in E243K and E243R, the P7 glutamate was replaced by positively charged lysine and arginine, ... the two enzymes distinguish the cofactors in different ways [3] In the present study, we have investigated, by means of site- directed mutagenesis and steady-state kinetics, the individual contribution ... however, coenzyme binding was tighter and therefore measurable and, in particular, gave measurable Kd values for binding of NADPH to both E243K and E243R, whereas the binding of NADH to these...
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Báo cáo khoa học: The phosphate site of trehalose phosphorylase from Schizophyllum communeprobed by site-directed mutagenesis and chemical rescue studies pptx
... the tasks fullled by Arg507 and Lys512 in the interaction network of active site residues during binding of phosphate and in catalysis Although we consider a-retaining glucosyl transfer via the ... residues for promoting a-retaining glycosyl transfer has not been well dened using mutagenesis and detailed kinetic analysis Results Analysis of kinetic consequences in R507A and K512A mutants ... original side chain in R507A and K512A, respectively A series of primary amines and derivatives of guanidine were therefore examined for their ability to restore phosphorylase activity in K512A and...
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Báo cáo khoa học: Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae Roles of Arg234 and Arg242 revealed by sequence analysis and site-directed mutagenesis doc
... analysis of phosphate and sulphate binding sites in proteins Estimation of propensities for binding and conservation of phosphate binding sites J Mol Biol 242, 321–329 22 Nandi, C.L., Singh, J & Thornton, ... arginine residues within the TOWER helix of StP at positions 234, 236 and 242 was interesting (Fig 2) Arginines are common components of protein interfaces and occur frequently at oxyanion-binding ... of arginine side chains in proteins Protein Eng 6, 247–259 23 Jones, S., Marin, A & Thornton, J.M (2000) Protein domain interfaces: characterization and comparison with oligomeric protein interfaces...
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