Tesis sobre el tema "Mutagenesi sito specifica"

CD33 is a myeloid cell surface marker absent on normal hematopoietic stem cells and normal tissues but present on leukemic blasts in 90% of adult and paediatric acute myeloid leukaemia (AML) cases. By virtue of its expression pattern and its ability to be rapidly internalized after antibody binding, CD33 has become an attractive target for new immunotherapeutic approaches to treat AML. In this study two immunoconjugates were constructed to contain a humanised single-chain fragment variable antibody (scFv) against CD33 in order to create new antibody-derived therapeutics for AML. The first immunoconjugate was developed to provide targeted delivery of siRNAs as death effectors into leukemic cells. To this purpose, a CD33-specific scFv, modified to include a Cys residue at its C-terminal end (scFvCD33-Cys), was coupled through a disulphide bridge to a nona-d-arginine (9R) peptide carrying a free Cys to the N-terminal. The scFvCD33-9R was able to completely bind siRNAs at a protein to nucleic acid ratio of about 10:1, as confirmed by electrophoretic gel mobility-shift assay. The conjugate was unable to efficiently transduce cytotoxic siRNA (siTox) into the human myeloid cell line U937. We observed slight reductions in cell viability, with a reduction of 25% in comparison to the control group only at high concentration of siTox (300 nM). The second immunoconjugate was constructed by coupling the scFvCD33-Cys to the type 1 ribosome inactivating protein Dianthin 30 (DIA30) through a chemical linking The resulting immunotoxin scFvCD33-DIA30 caused the rapid arrest of protein synthesis, inducing apoptosis and leading ultimately to cell death. In vitro dose-dependent cytotoxicity assays demonstrated that scFvCD33-DIA30 was specifically toxic to CD33-positive cell U937. The concentration needed to reach 50 % of maximum killing efficiency (EC50) was approximately 0.3 nM. The pronounced antigen-restricted cytotoxicity of this novel agent makes it a candidate for further evaluation of its therapeutic potential.

CD33 is a myeloid cell surface marker absent on normal hematopoietic stem cells and normal tissues but present on leukemic blasts in 90% of adult and paediatric acute myeloid leukaemia (AML) cases. By virtue of its expression pattern and its ability to be rapidly internalized after antibody binding, CD33 has become an attractive target for new immunotherapeutic approaches to treat AML. In this study two immunoconjugates were constructed to contain a humanised single-chain fragment variable antibody (scFv) against CD33 in order to create new antibody-derived therapeutics for AML. The first immunoconjugate was developed to provide targeted delivery of siRNAs as death effectors into leukemic cells. To this purpose, a CD33-specific scFv, modified to include a Cys residue at its C-terminal end (scFvCD33-Cys), was coupled through a disulphide bridge to a nona-d-arginine (9R) peptide carrying a free Cys to the N-terminal. The scFvCD33-9R was able to completely bind siRNAs at a protein to nucleic acid ratio of about 10:1, as confirmed by electrophoretic gel mobility-shift assay. The conjugate was unable to efficiently transduce cytotoxic siRNA (siTox) into the human myeloid cell line U937. We observed slight reductions in cell viability, with a reduction of 25% in comparison to the control group only at high concentration of siTox (300 nM). The second immunoconjugate was constructed by coupling the scFvCD33-Cys to the type 1 ribosome inactivating protein Dianthin 30 (DIA30) through a chemical linking The resulting immunotoxin scFvCD33-DIA30 caused the rapid arrest of protein synthesis, inducing apoptosis and leading ultimately to cell death. In vitro dose-dependent cytotoxicity assays demonstrated that scFvCD33-DIA30 was specifically toxic to CD33-positive cell U937. The concentration needed to reach 50 % of maximum killing efficiency (EC50) was approximately 0.3 nM. The pronounced antigen-restricted cytotoxicity of this novel agent makes it a candidate for further evaluation of its therapeutic potential.

Accessory hydrogen uptake genes have been identified in a region of the Azotobacter chroococcum genome about 5 kb downstream of the hydrogenase structural genes (hupSL). DNA sequencing has revealed six genes (hupABYCDE) in this region. These genes are probably transcribed in the same direction as hupSL but are probably in a different operon. Mutational analysis had shown that disruption of the hupB, hupY, hupD and hupE genes gives a Hup$ sp-$ phenotype. In the present work additional mutational analysis, using Tn5, a Tn5 -derivative containing a promoterless lacZ gene, and a kanamycin resistance gene, confirms the direction of transcription and the separate nature of the hupABYCDE operon, and extends the region known to be necessary for Hup activity to hupA and possibly to 1.6 kb upstream of hupA.

V-ATPases are enzymes found in all eukaryotic cells. They are organized into a peripheral membrane complex (V1) and an integral membrane complex (V0). VI is responsible for ATP hydrolysis and generates the energy used by Vo to pump protons from the cytosol into the vacuole. Subunit d is a component of Vo possibly located at the interface between V 1 and V. in the V-ATPase complex. We hypothesize that subunit d could be involved in the structural and functional coupling of VI and Vo. This was tested by generating point mutations along the open reading frame of subunit d from yeast. The mutations F94A, H128A, D173A, D217A, D261A, E317A, W325A, E328A and C329A, all in conserved regions of the protein sequence, were characterized by examining their growth phenotype and by assessing their ATPase specific activity, proton transport and V1Vo assembly in purified vacuolar membranes. The mutations E317A, W325A, E328A and C329A had reduced ATPase and proton transport activities. In addition, V1Vo assembly was compromised by the mutation W325A. Our results suggest that residues at the carboxyl-end of subunit d are important for ATPase activity, proton pumping and V1Vo assembly at the membrane.
Department of Chemistry

Molecular chaperones help many proteins in the cell reach their native conformation. The mechanism with which they do this has been studied extensively, but has not been entirely elucidated. This work is a continuation of the study done by Laila Villebeck et al. (2007) on the conformational rearrangements in the eukaryotic protein actin in interaction with the eukaryotic chaperone TRiC. In this study the intentions were to analyze the protein MreB, a prokaryotic homologue to actin, when interacting with the prokaryotic chaperone GroEL. The purpose was to investigate if the mechanisms of GroEL and TRiC are similar. The analysis of the conformation of MreB was to be made through calculations of fluorescence resonance energy transfer (FRET) between two positions in MreB labeled with fluorescein. A MreB mutant was made through site-specific mutagenesis to enable labeling at a specific position. Another single mutant and a corresponding double mutant needed for these measurements were avaliable from earlier studies. The results from fluorescence measurements on these mutants indicated that the degree of labeling was insufficient for accurate determination of FRET. Suggestions are made on improvements of the experimental approach for future studies.

Flavonoids are plant secondary metabolites that have significant biochemical and physiological roles. Biosynthesis of these compounds involves several modifications, most predominantly glucosylation, which is catalyzed by glucosyltransferases (GTs). A signature amino acid sequence, the PSPG box, is used to identify putative clones and has been shown to be involved in UDP-glucose binding. Site-directed mutagenesis is used to answer questions regarding the structure and function of this family of enzymes, particularly what allows some GTs to be more selective towards some substrates than others. The grapefruit (Citrus paradisi) flavonol-3-O-glucosyltransferase (CpF3GT) is specific for flavonol substrates and will not glucosylate anthocyanidins. Comparison of the CpF3GT sequence with that of Vitis vinifera GT, which glucosylates both flavonols and anthocyanidins, provided the basis for the amino acid substitution of proline 145, alanine 374, and alanine 375 in CpF3GT to threonine, aspartate, and glycine, respectively, to test the affect on GT’s affinity for flavonoid substrates.

Point mutations were engineered into the sequence of the multispecific DNA methyltransferase (Mtase) M. SPRI in motif IX, in order to mimic the corresponding motif IX of mono-specific Mtase. A similar approach was adopted to modify the sequence of the monospecific enzyme M. HhaI in motifs IX and X based on the available structure and as a consequence the enzyme regained methylation potential. It was thought that these changes might be sufficient to enable functional exchange of the target recognition domains (TRDs) between a mono- and a multispecific enzyme. However, insertion of various segments of TRD region from M. SPRI into the M. HhaI was not successful (Chapter 4). To establish whether mono- and multispecific Mtases are incompatible in terms of sequence exchanges, a systematic "swapping" of motifs was carried out (Chapter 5). These experiments suggested that there are some enzyme-specific structural interactions between different subunits within each class of Mtases. In second half of this thesis a bacterial two-hybrid system based on the reversible assembly of an engineered form of M. SPRI was developed (Chapter 6). However the Mtase protein does not assemble into an active species until a DNA segment encoding a leucine zipper motif is fused to each of the two halves. Co-transformation of E. coli with the plasmids expressing the C-terminal and N-terminal domains respectively resulted in the abolition of colonies on double antibiotic plates, when an mcr strain was used as host. High performance liquid chromatography was used to estimate the extent of modification of plasmids indirectly. The extent of methylation at specific sequences within a plasmid molecule was readily detected by the corresponding differential susceptibility to digestion by specific restriction enzymes. Using this approach it proved possible to detect different levels of activity produced by wild type and mutant recombinant DNA Methyltransferases with sensitivity and in a semi quantitative manner. In order to analyse the biochemical properties of Mtase, I have developed an in vitro translation-modification assay. Binary studies with the mutants (from Chapter 3 and 5) showed that there were no detectable sequence-specific recognition differences between these enzymes. Taken together, these results suggest that motif IX plays a role in general stabilisation of the enzyme core structure and has a less significant role in DNA recognition.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003.
Includes bibliographical references (leaves 82-99). Also available in electronic version. Access restricted to campus users.

A primary aim of this study was to establish and utilise the supF assay to investigate the mutagenicity of the cancer drug tamoxifen in target endometrial cells. In particular, the supF assay was employed to ascertain the mutations caused by two model reactive intermediates of tamoxifen, alpha-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide (4-OHtamQM) in methylated pSP189 plasmid. The plasmid was methylated in vitro in order to allow application of the LwPy53 algorithm which enables in silico prediction of the G→T mutation distribution along the human p53 gene, using alpha-acetoxytamoxifen and 4-OHtamQM-induced mutation data from the supF assay.;Relative mutation frequencies increased proportionally with the adduct level for alpha-acetoxytamoxifen, up to ∼15 times the background frequency, while 4-OHtamQM failed to raise the mutation frequency above that of the untreated control. The majority of mutations in alpha-acetoxytamoxifen-treated plasmid were GC →TA transversions, while GC→AT transitions predominated in both 4-OHtamQM-treated plasmid and the untreated control. Based on the p53 G→T transversion predictions, alpha-acetoxytamoxifen induced damage resulted in two hotspots at positions 244 and 273; these mutations might be expected to occur in tamoxifen associated endometrial tumours if tamoxifen adducts play a role in cancer development.;The second part of the thesis was devoted to the development and validation of a novel site-specific assay. The assay, which is a site-specific version of the supF assay, was validated and utilized to investigate the mutagenic potential and types of mutations caused by individual O 6-MeG adducts situated in intact double stranded or gapped plasmids, within two different sequence contexts, in both E. coli and human cells.

Toksino-antitoksino (TA) sistemos – tai poros viename operone esančių bakterijų ir archėjų genų, kurių vienas koduoja toksišką baltymą, o antras – jį neutralizuojantį baltymą-antitoksiną. Tol, kol ląstelėje gaminamas pakankamas abiejų baltymų kiekis, antitoksinas jungiasi su toksinu ir jį išaktyvina. Tačiau, esant nepalankioms aplinkos sąlygoms, labilesnis antitoksinas suardomas aktyvintų proteazių, o likęs laisvas stabilesnis toksinas slopina gyvybiškai svarbius ląstelinius procesus – baltymų arba DNR biosintezę, dėl ko stabdomas ląstelių augimas arba jos žūva. Escherichia coli chromosomoje aprašyta daugiau nei dešimt TA sistemų, kurių viena yra dinJ-yafQ, apie kurią žinoma labai nedaug. Anksčiau laboratorijoje atliktuose darbuose nustatyta, kad dinJ-yafQ koduoja transliaciją slopinantį toksiną YafQ, o DinJ ir YafQ baltymai sudaro stiprų baltymų kompleksą, slopinantį YafQ toksišką poveikį. Kol kas nieko nėra žinoma apie YafQ molekulės sritis, svarbias sąveikai su antitoksinu DinJ. Šiame darbe sekai atrankios mutagenezės metodu buvo tirtos YafQ baltymo sritys, svarbios sąveikai su „savuoju“ toksinu DinJ. TA sistemoms būdinga savo operono transkripcijos autoreguliacija. DNR sulėtinimo gelyje eksperimentais parodėme atrankią DNR ir antitoksino DinJ bei DinJ-YafQ baltymų komplekso sąveiką. Laisvas antitoksinas DinJ silpniau sąveikauja su DNR nei būdamas komplekse su YafQ, o sąveikai su DNR svarbi DinJ baltymo N galinė dalis. Iš dviejų dinJ-yafQ operono promotoriaus srityje... [toliau žr. visą tekstą]
Prokaryotic toxin antitoxin systems consist of two adjacent genes, where one encodes a stable toxin harmful to essential cellular processes (translation or DNA synthesis), and the other a labile antitoxin, capable of blocking the toxin's activity by binding into stable protein complex. TA systems are proposed to be involved in bacterial adaptation to stress conditions by modulating the level of essential biological processes. There are at least ten characterized chromosome-encoded TA loci in Escherichia coli. The dinJ-yafQ operon codes for YafQ toxin which is neutralized by its cognate antitoxin, DinJ. YafQ is known to inhibit translation in vivo and belongs to the RelE toxin family of toxin ribonucleases. By using site-specific mutagenesis of YafQ, we have investigated the protein regions important for its interaction with DinJ antitoxin. Transcriptional autoregulation has been reported for members of all known TA gene families and appears to be general characteristic of regulation of TA loci. In this work electrophoretic mobility shift assay was used to investigate the interaction between the antitoxin DinJ and DinJ-YafQ complex and dinJ-yafQ operon promoter DNA. Antitoxin DinJ in the complex with YafQ had an enhanced DNA-binding affinity compared to free DinJ. N-terminal domain of antitoxin is crucial for interaction with DNA. Bioinformatic analysis of dinJ-yafQ operon promoter region revealed several palindromic DNA islands and their importance for interaction with DinJ... [to full text]

Flavonoids are secondary metabolites that are important in plant defense, protection, and human health. Most naturally-occurring flavonoids are found in glucosylated forms. Glucosyltransferases catalyze the transfer of glucose from high-energy sugar donors to an acceptor molecule. The grapefruit flavonol-specific 3-O-glucosyltransferase (F3-O-GT) is highly substrate and regio-specific. The goal of this research is to unravel the amino acid residues responsible for the grapefruit enzyme’s rigid specificity, while attempting to alter the regiospecific glucosylation pattern through site-directed mutagenesis and homology modeling. This research tested the hypothesis that substitution of potential key amino acid residues within the grapefruit Cp-F3-O-GT with position equivalent residues within F7-O-GTs would alter the 3-O-glucosylation of the enzyme. Results reveal that specific single point mutations of residues are capable of abolishing enzymatic activity. Recombinant mutant G392E retained activity and showed an increased affinity for kaempferol relative to the wild-type; however, the rigid regiospecific glucosylation pattern of the enzyme was retained.

Plasma membrane transport proteins play a crucial role in all cells by conferring to the cell surface a selective permeability to a wide range of ions and small molecules. The activity of these transporters is often regulated by controlling their amount at the plasma membrane, via intracellular trafficking. The recent boom in the numbers of crystallized transporters shows that many of them that belong to different functional families with little sequence similarity adopt the same structural fold implying a conserved transport mechanism. These proteins belong to the APC (Amino acid-Polyamine-organoCation) superfamily and their fold is typified by the bacterial leucine transporter LeuT. This LeuT fold is characterized by inverted structural repeats of 5 transmembrane domains that harbor the central substrate-binding site and a pseudo-symmetry axis parallel to the membrane. The yeast Saccharomyces cerevisiae possesses about 16 amino acid permeases (yAAPs) that belong to the APC superfamily and that display various substrate specificity ranges and affinities. Topological, mutational analysis and in silico data indicate that yAAPS adopt the LeuT fold.

In this work we combined computational modeling and yeast genetics to study substrate binding by yAAPs and the endocytosis of these transporters in response to substrate transport. In the first part of this work, we analyzed the selective recognition of arginine by the yeast specific arginine permease, Can1. We constructed three-dimensional models of Can1 using as a template the recently resolved structure of AdiC, the bacterial arginine:agmatine antiporter, which is also a member of the APC superfamily. By comparison of the binding pockets of Can1 and Lyp1, the yeast specific lysine permease, we identified key residues that are involved in the recognition of the main and side chains of arginine. We first showed that the network of interactions of arginine in Can1 is similar to that of AdiC, and that the selective recognition of arginine is mediated by two residues: Asn 176 and Thr 456. Substituting these residues by their corresponding residues in Lyp1 converted Can1 into a specific lysine permease. In the second part of this work, we studied the regulation of two permeases, Can1 and the yeast general amino acid permease, Gap1. In the presence of their substrates, Gap1 and Can1 undergo ubiquitin-dependent endocytosis and targeting to the vacuolar lumen for degradation. We showed that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. By permease structural modeling, mutagenesis, and kinetic parameter analysis, we showed that Gap1 and Can1 need to switch to an intermediary conformational state and persist a minimal time in this state after binding the substrate to trigger their endocytosis. This down-regulation depends on the Rsp5 ubiquitin ligase and involves the recruitment of arrestin-like adaptors, resulting in the ubiquitylation and endocytosis of the permease.

Our work shows the importance of the structural analysis of yAAPs to get further insight into the different aspects of their function and regulation. We validate the use of a bacterial APC transporter, AdiC, to construct three-dimensional models of yAAPs that can be used to guide experimental analyses and to provide a molecular framework for data interpretation. Our results contribute to a better understating of the recognition mode of amino acids by their permeases, and the regulation of this transport in response to substrate binding.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

碩士
國立臺灣科技大學
化學工程系
91
Abstract The object of this study was to construct a site-specific random mutant library and setup a high throughput screening method to study the stability roles of residues in the catalytic cleft of N-carbamoyl-D-amino acid amidohydrolase, DCase. Based on the crystallography structure of DCase, six catalytic-cleft residues, His144, Glu146, Arg175, Arg176, Phe53, and Phe54 in DCaseH, a recombinant DCase carrying a 6 × His tag in C-terminus were substituted with other amino acids by both PCR and cassette random mutageneses. In the mean time, high throughput screening process was setup by employing a robotic colony picker, Qpix, and colorimetric enzyme assay of DCase activity. About 18000 colonies were picked out and analyzed with an aqueous colorimetric assay in the first round of screening, resulting in 250 active clones. In the second round of screening, a triple mutant, F54L\H144T\E146S, with a better thermostability than that of wild-type DCaseH was selected using a membrane-colorimetric assay. To investigate the individual stability role of each mutagenized residue, three single mutants, F54L, H144T, and E146S, were constructed by site-directed mutagenese. The production level and purification efficiency of all four mutants were similar to those of wild-type DCaseH, while their catalytic activities were significantly lower. Nevertheless, the thermostabilities of E146S and the triple mutant were clearly higher than that of the wild-type one, suggesting that the Glu146→Ser substitution enhanced the thermostability of DCaseH. Furthermore, the secondary and tertiary structure analyses by circular dichroism and fluorescence spectroscopies revealed that the structures of F54L and the triple mutant were significantly affected, suggesting that the Phe54→Leu mutation caused the structure perturbation. Finally, the superposition of the molecularly modeled structures of the DCaseH mutant with that of the wild-type one did not reveal any difference, presumably because an improper molecule-modeling procedure was used.

碩士
元培科學技術學院
生物技術研究所
93
Delonix regia is a Leguminosae that belongs to the Caesalpininidae subfamily. The Delonix regia trypsin inhibitor (DrTI) was a serine protease inhibitor isolated from its seeds. DrTI was purified sequentially by ammonium sulfate precipitation (30%-60%), gel filtration, DEAE-52 ion exchange chromatography and affinity chromatography. A molecular weight of 22 kDa was estimated by SDS-PAGE. SDS-PAGE showed that DrTI consisted of a single polypeptide chain. The DrTI was found to be a thermostable Kunitz type TI. Proteinase inhibitors may also have a role in the treatment of human pathologies such as inflammation, hemorrhage, and cancer. In this study, degenerate primers were designed based on all possible sequences of the N-terminal and C-terminal region of Delonix regia trypsin inhibitor (DrTI). 561 bp of PCR product was amplified using the above degenerate primers, and genomic DNA and cDNA of Delonix regia as a template. The amplified PCR products were cloned and sequenced. DNA sequence analysis of cDNA and genomic clones of DrTI have cloned two clones, DrTI-A and DrTI-B, and demonstrated genomic clone without intervening sequences in the coding region. The amino acid sequence deduced from the DrTI genomic or cDNA clones agreed with that identified via amino acid sequencing analysis, except that two amino acid residues, Serine and lysine, existed between the residues Lys141and Ser142. DrTI ORF was then amplified and cloned in-frame in pQE-60 and overexpressed in Escherichia coli to yield a histidine-tagged recombinant protein with a calculated molecular mass of about 23 kDa. Both the reDrTI and native-DrTI protein exhibit a strong and identical inhibitory effect on trypsin activity. Site-directed mutagenesis was performed using PCR to identify what the active site of the DrTI amino acid residues. We find the reDrTIE68K mutant lost some inhibitory effect on trypsin activity, and reDrTIE68L mutant almost lost its activity, but reDrTIK72E mutant retained most of its inhibitor activity.The C139G mutant lost its inhibitor activity while the C44G mutant did not. This suggests that the Glu68 is located at the reactive site of DrTI. It’s also demonstrated that the trypsin inhibitor activity of reDrTIC44G mutant was similar to native DrTI, whereas the reDrTIC139G mutant lost its trypsin inhibitor activity. It suggests that the second-disulfide bond (Cys139-Cys149) is important for the trypsin inhibitor activity. We also demonstrated that DrTI could induce Jurkat cell apoptosis by MTT test and DNA ladder analysis. These experiments suggest that the DrTI may play an important role in the apoptosis mechanism. Keywords: Delonix regia trypsin inhibitor (DrTI), site-specific mutagenesis, disulfide bond, Jurkat cell.

碩士
國立臺灣海洋大學
生物科技研究所
96
The eurythermic Cyprinus carpio (common Carp) can live from 35 ℃ to 10 ℃. Its muscle type creatine kinase (CK, EC 2.7.2.3) is one of the most important energy metabolism enzymes which can maintain their enzyme requirements for survival at varying temperature. We have previously cloned two muscle-specific sub-isoform of CK from Carp, designated M1-CK and M3-CK. The M1-CK has higher specific activity than M3-CK at pH7.1 ~ 8.0 and 0 ℃ ~ 50 ℃. CK becomes in dimer form and the first six residues at N-terminal of CK influence its specific activity and subunit cohesion. There is only 16.7 % similarity at the first 6 residues at the N-terminal between M1-CK and M3-CK, and the specific activity of M3-CK is much lower than M1-CK. Thus, in this study, site-directed mutagenesis was used to mutate the amino acids that are at the N-terminal of M3-CK, then hope to find the specific amino acid which is important to subunit cohesion and thus influences its specific activity. Pro2 and His6 were found to have effects on its specific activity, the ability of the association with substrate, and thermal stability.

碩士
國防醫學院
生物化學研究所
89
Pigeon liver malic enzyme [EC1.1.1.40] is a homotetramer with each subunit of molecular weight 62 kDa. This enzyme is a bifunctional enzyme that catalyzes the reversible oxidative decarboxylation of L-malate, yielding pyruvate and CO2, with a concomitant conversion of coenzyme NADP+ to NADPH. The three dimensional structures of pigeon malic enzyme was constructed by the computer modeling. There are six threonine residues closed to the active site. These six threonine residues were mutated to alanine residues by alanine scanning site-directed mutagenesis. Mutation of threonine residues at positions 92 and 399 showed significant effect on the Km value for malate. Replacement of these two threonine residues to valine or serine and the double mutant T(92,399)A were constructed. Initial velocity studies demonstrated that both hydroxyl group and correct carbon chain are essential for malate binding. Inhibition studies of malate analogs, DL-a-hydroxybutyric acid and DL-b-hydroxybutyric acid, were also performed. In conclusion, we suggest that Thr 92 may interact with C4 carboxyl group of malate through a hydrogen bond.

L'azote est l'un des éléments les plus essentiels dans le monde pour les êtres vivants, car il est essentiel pour la production des éléments de base de la cellule, les acides aminés, les acides nucléiques et les autres constituants cellulaires. L’atmosphère est composé de 78% d'azote gazeux, une source d'azote inutilisable par la plupart des organismes à l'exception de ceux qui possèdent l’enzyme nitrogénase, tels que les bactéries diazotrophique. Ces micro-organismes sont capables de convertir l'azote atmosphérique en ammoniac (NH3), qui est l'une des sources d'azote les plus préférables. Cette réaction exigeant l’ATP, appelée fixation de l'azote, est catalysée par une enzyme, nitrogénase, qui est l'enzyme la plus importante dans le cycle de l'azote. Certaines protéines sont des régulateurs potentiels de la synthèse de la nitrogénase et de son activité; AmtB, DraT, DraG, les protéines PII, etc.. Dans cette thèse, j'ai effectué diverses expériences afin de mieux comprendre leurs rôles détailés dans Rhodobacter capsulatus. La protéine membranaire AmtB, très répandue chez les archaea, les bactéries et les eucaryotes, est un membre de la famille MEP / Amt / Rh. Les protéines AmtB sont des transporteurs d'ammonium, importateurs d'ammonium externe, et ont également été suggéré d’agir comme des senseurs d'ammonium. Il a été montré que l’AmtB de Rhodobacter capsulatus fonctionne comme un capteur pour détecter la présence d'ammonium externe pour réguler la nitrogénase. La nitrogénase est constituée de deux métalloprotéines nommées MoFe-protéine et Fe-protéine. L'addition d'ammoniaque à une culture R. capsulatus conduit à une série de réactions qui mènent à la désactivation de la nitrogénase, appelé "nitrogénase switch-off". Une réaction critique dans ce processus est l’ajout d’un groupe ADP-ribose à la Fe-protéine par DraT. L'entrée de l'ammoniac dans la cellule à travers le pore AmtB est contrôlée par la séquestration de GlnK. GlnK est une protéine PII et les protéines PII sont des protéines centrales dans la régulation du métabolisme de l'azote. Non seulement la séquestration de GlnK par AmtB est importante dans la régulation nitrogénase, mais la liaison de l'ammonium par AmtB ou de son transport partiel est également nécessaire. Les complexes AmtB-GlnK sont supposés de lier DraG, l’enzyme responsable pour enlever l'ADP-ribose ajouté à la nitrogénase par DraT, ainsi formant un complexe ternaire. Dans cette thèse certains détails du mécanisme de transduction du signal et de transport d'ammonium ont été examinés par la génération et la caractérisation d’un mutant dirigé, RCZC, (D335A). La capacité de ce mutant, ainsi que des mutants construits précédemment, RCIA1 (D338A), RCIA2 (G344C), RCIA3 (H193E) et RCIA4 (W237A), d’effectuer le « switch-off » de la nitrogénase a été mesurée par chromatographie en phase gazeuse. Les résultats ont révélé que tous les résidus d'acides aminés ci-dessus ont un rôle essentiel dans la régulation de la nitrogénase. L’immunobuvardage a également été effectués afin de vérifier la présence de la Fe-protéine l'ADP-ribosylée. D335, D388 et W237 semblent être cruciales pour l’ADP-ribosylation, puisque les mutants RCZC, RCIA1 et RCIA4 n'a pas montré de l’ADP-ribosylation de la Fe-protéine. En outre, même si une légère ADP-ribosylation a été observée pour RCIA2 (G344C), nous le considérons comme un résidu d'acide aminé important dans la régulation de la nitrogénase. D’un autre coté, le mutant RCIA3 (H193E) a montré une ADP-ribosylation de la Fe-protéine après un choc d'ammonium, par conséquent, il ne semble pas jouer un rôle important dans l’ADP-ribosylation. Par ailleurs R. capsulatus possède une deuxième Amt appelé AmtY, qui, contrairement à AmtB, ne semble pas avoir des rôles spécifiques. Afin de découvrir ses fonctionnalités, AmtY a été surexprimée dans une souche d’E. coli manquant l’AmtB (GT1001 pRSG1) (réalisée précédemment par d'autres membres du laboratoire) et la formation des complexes AmtY-GlnK en réponse à l'addition d’ammoniac a été examinée. Il a été montré que même si AmtY est en mesure de transporter l'ammoniac lorsqu'il est exprimé dans E. coli, elle ne peut pass’ associer à GlnK en réponse à NH4 +.
Nitrogen is one of the most vital elements in the world for living creatures since it is essential for the production of the basic building blocks of the cell; amino acids, nucleic acids and other cellular constituents. The atmosphere is 78% nitrogen gas (N2), a source of nitrogen unusable by most organisms except for those possessing the enzyme nitrogenase, such as diazotrophic bacteria species. These microorganisms are capable of converting atmospheric nitrogen to ammonia (NH3), which is one of the most preferable nitrogen sources. This ATP demanding reaction, called nitrogen fixation, is catalysed by the nitrogenase enzyme, which is the most important enzyme in the nitrogen cycle. Some proteins are potential regulators of nitrogenase synthesis and activity; AmtB, DraT, DraG, PII proteins and etc. In this thesis I performed various experiments in order to better understand their roles in Rhodobacter capsulatus, in more detail. The membrane protein AmtB, which is widespread among archaea, bacteria and eukaryotes, is a member of the MEP/Amt/Rh family. The AmtB proteins are ammonium transporters, taking up external ammonium, and have also been suggested to sense the presence of ammonium. It has been shown that in Rhodobacter capsulatus AmtB functions as a sensor for the presence of external ammonium in order to regulate nitrogenase. Nitrogenase consists of two metalloprotein components named MoFe-protein and Fe-protein. The addition of ammonium to R. capsulatus culture medium leads to a series of reactions which result in the deactivation of nitrogenase, called “nitrogenase switch-off”. A critical reaction in this process is one in which DraT adds an ADP-ribose group to the Fe-protein of nitrogenase. The entrance of ammonia through the AmtB pore is regulated by GlnK sequestration. GlnK is a PII protein and PII proteins are one of the central proteins in the regulation of nitrogen metabolism. Not only is GlnK-AmtB sequestration important in nitrogenase regulation, but binding of ammonium by AmtB or its partial transport is also necessary. AmtB-GlnK complexes are thought to bind DraG, which is responsible for removing the ADP-ribose that DraT adds to nitrogenase, to form a ternary complex. In this thesis details of the signal transduction mechanism and ammonium transport were examined by generating and characterizing RCZC, a (D335A) site- directed mutant of AmtB. The ability of this mutant, as well as previously constructed mutants RCIA1 (D338A), RCIA2 (G344C), RCIA3 (H193E) and RCIA4 (W237A), to “switch-off” nitrogenase activity was measured by gas chromatography. The results revealed that all the above amino acid residues have critical roles in nitrogenase regulation. Immunoblotting was also carried out to check the presence of ADP-ribosylated Fe-protein. D335, D388 and W237 seem to be crucial for NifH ADP-ribosylation, since their mutants (RCZC, RCIA1 and RCIA4 respectively) didn't show ADP-ribosylation on Fe-protein. In addition, although a slight ADP-ribosylation was observed for RCIA2 (G344C) we still consider it as an important amino acid residue in this matter whereas the remaining mutant RCIA3 (H193E) showed Fe-protein ADP-ribossylation after an ammonium shock, therefore it doesn't seem to be important in NifH ADP-ribosylation. In addition R. capsulatus possesses a second Amt called AmtY, which in contrast to AmtB, doesn't appear to have any specific roles. In order to find out its functionality, AmtY was overexpressed in an E. coli strain lacking AmtB (GT1001 pRSG1) (which was carried out previously by other lab members) and AmtY-GlnK complex formation in response to ammonium addition was examined. It was shown that even though AmtY is able to take up ammonia when expressed in E. coli it fails to associate with GlnK in response to NH4+.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1985.
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Nitrilases are enzymes which catalyze the hydrolysis of a nitrile into the corresponding carboxylic acid and ammonia. These enzymes are potentially applicable in biocatalysis and bioremediation because of their advantages over the conventional (chemical) methods of nitrile hydrolysis (lower demand for energy, safety, simplicity, high yields, selectivity). In this work, genome mining was used to search for the sequences of hypothetical nitrilases from filamentous fungi. The amino acid sequences of previously characterized fungal nitrilases were used as the templates. Then the new synthetic genes together with other genes from our nitrilase library were expressed in E. coli and the substrate specificities of the enzymes thus produced were compared. Significant attention was focused on the relationships between the sequence of the enzyme and its substrate specificity. The arylacetonitrilases from Arthroderma benhamiae (NitAb) and Nectria haematococca (NitNh) were purified and characterized. Their substrate specificities, kinetic parameters, pH and temperature profiles and subunit and holoenzyme size were assessed. NitAb and NitNh together with other recombinant fungal nitrilases were employed in the hydrolysis of high concentrations of (R,S)-mandelonitrile in a batch or fed-batch mode. Nitrilase from...

碩士
國防醫學院
生物化學研究所
84
Malic enzyme was rapidly inactivated by Fe2+-ascorbate at neutral pH. The inactivated enzyme was subsequently cleaved at the chemical bond between Asp258 and Ile259 [Wei, C. H., Chou, W. Y., Huang, S. M., Lin, C. C.,and Chang, G. G. (1994) Biochemistry, 33, 7931-7936], which was identified as the Mn(II) binding site by site-specific mutagenesis [Wei, C. H., Chou, W. Y., and Chang, G. G. (1995) Biochemistry, 34, 7949-7954]. At acidic pH, however, Fe2+ was found to be ineffective in oxidative modification o f the enzyme. Nevertheless, Cu2+ still caused enzyme inactivation and cleaved the enzyme at Asp141-Gly142, Asp194-Pro195, or Asp464-Asp465, which suggest that Asp141, Asp194, and Asp464 are also the coordination sites for the metal-binding of malic enzyme [Chou, W. Y., Tsai, W. P., Lin, C. C., and Chang , G. G. (1995) J. Biol. Chem., 270, 25935-25941]. In this study, I prepare three mutant malic enzymes D141N, D194N and D464N. These recombinant proteins were successfully expressed in a bacterial expressio n system (pET-15b), which introduced three amino acid residues Met-Asp-Ser attached at the N- terminus. This attachment, however, was assumed not affecting my comparison of the properties of WT with those of the mutants. I observed no differences of the kinetic parameters between the WT, D194N and D464N. The Asp194 and Asp464 thus are not directly involved in the binding or catalytic reaction of the enzyme. The KmMal for D141N, however, was increased by approximately 3-fold. The KmMn for D141N mutant in creased approximately 10-fold. Furthermore, D141N showed no substrate inhibition phenomenon, which was observed for WT, D194N and D464N. In combination, the catalytic efficiency kcat/(KmMn(KmMalate(KmNADP) of D141N decreased approximately by 20-fold as compared to WT. Cu2+ was found to be a competitive inhibitor versus Mn2+ for all recombinant enzymes.

博士
國立臺灣大學
生化學研究所
81
PART I:相思樹胰蛋白脢抑制劑(ACTI)由A鏈及B鏈以一對雙硫鍵連結所組 成 ,A鏈含136個氨基酸,B鏈含39個氨基酸,A分子量共19.4KDa,屬於 Kunitz-typ trypsin抑制劑.合成可能的相思樹胰蛋白脢抑制劑A鏈的氨端 和B鏈的C端氨基酸的Oligonucleotides當作Primers,萃取尚未成熟種子的 cDNA當作模板,利用PCR的技術,選殖了ACTI的cDNA序列,轉譯成176個氨基 酸,發現比蛋白序列多出一個氨基酸,Ser137,其位於A鏈與B鏈連結的位置, 成熟的ACTI需藉一特異的酵素進行Post-translation processing將它切 除。將ACTI的Coding region崁入pGEX-2T質體,置於大腸桿菌中表現,沒有 經過Post-translation processing的融合蛋白質和重組ACTI皆具有可抑 制胰蛋白脢抑制劑活性。 Lys64可能是活性位置,所以利用Site- Specific mutagenesis將Lys64突變成Ile和Arg,K64I其活性完全喪失,但 K64R其活性幾乎不變,顯示Lys64-Ile 65是其活性位置,屬於離氨酸型的胰 蛋白脢抑制劑,且活性位置上離氨酸和精氨酸可互換而不影響其活性. ACTI含有兩對雙硫鍵,分別破壞其雙硫鍵的形成,結果位於A鏈內這對雙硫 鍵(Cys40-Cys86)對ACTI的活性影響很小,但連結A鏈與B鏈的這對雙硫鍵 Cys133-Cys141)破壞後,整個分子結構改變且活性完全喪失. PART II:雞 母珠毒蛋白是由A鏈及B鏈以一對雙硫鍵連結所組成,B鏈具有辨識細胞表面 受體的能力,A鏈具有N-glycosidase的活性,當進入細胞內,A鏈能水解真核 細胞核醣體上28S rRNA第4324位置上Adenine 的醣甘犍,而抑制蛋白質生 合成.雞母珠毒蛋白具有多種Isoforms,對蛋白質生合成抑制能力不相同. 利用PCR技術,選殖了三株異雞母珠毒蛋白cDNAs,依據氨基酸序列,物理化 學,和免疫反應,推測這三株異雞母珠毒蛋白是Abrin-a, Abrin-b和Abrin- d.將異雞母珠毒蛋白的A鏈嵌入pGEX-2T質體,置於大腸桿菌中表現,分析對 無細胞蛋白質生合成抑制活性或N-glycosidase活性,對無細胞蛋白質生合 成抑制活性分析結果,重組Abrin-a和Abrin-d的A鏈活性與原始Abrin-a A 鏈相似,但重組Abrin-b A鏈活性約低3倍,而N-glycosidase活性,重組 Abrin-b A鏈比另外兩種重組Abrin A鏈約低10倍.將Abrin-a A鏈可能是活 性位置的Glu164突變成Ala，Arg167突變成Leu或兩者皆突變,結果 ,在抑 制蛋白質生合成活性分析,E164A與R167L分別降低了25倍和625倍,而 E164 A.R167L更降低了1250倍,而N-glycisidase分析,與抑制蛋白質生合成之結 果相似.所以Glu164可能僅是穩定酵素與受質結和的結構，而Arg167直接 與催化作用有關. Part I:ACTI(Acacia confusa trypsin inhibitor)is one of the Kunitz -type inhibitors.The native form is composed of two different po- lypeptide: the A and B-chains, 136 amino acids and 39 amino acids residues,respectively.The molecular weight of ACTI is 19.4 kDa. The deduced amino acid sequence agreed with that determined by the peptide analysis except an extra amino acid residue,Ser, was found at the junction of A and B chain,which was removed by post- translation processing with specific protease(s). A recombinant plasmid containing the coding regions for ACTI has been construct ed and expressed in Escherichia coli cells,as a fusion protein be tween ACTI and glutathione S-transferase(GST).Both the reACTI and fusion protein have a strong inhibitory effect on trypsin activi- ty without post-translational proteolysis.We converted the Lys64 residue into Ile or Arg residue by using site-specific mutagene- sis to identify the active site of ACTI.The reACTIK64 I mutant lo- st its inhibitory effect on trypsin activity but the reACTIK64R mutant almost did not change its activity.It indicates that Lys64 is located at the reactive site of ACTI, and ACTI is one of lysine -type trypsin inhibitors. Lys or Arg at position of active site can be replaced each other,and it still maintains its fully inhi- bitory effect on trypsin activity.ACTI contains two disulfide bo- nds;one is an intradisulfide bond(Cys40-Cys86)locating in A chain and another is an interdisulfide bond(Cys133-Cys141)linking A and B chains. The disulfide bond was removed by converting Cys to Gly by site- specific mutagenesis. The results demonstrated that the trypsin inhibitory activity of reACTIC40G mutant was almost iden- tical to that of native ACTI,wheras the reACTIC133G mutant lost its trypsin inhibitory activity completely caused by the change of molecular conformation.It suggests that the interdisulfide bo- nd(Cys133-Cys141)is indispensible for the trypsin inhibitory act- ivity.

[ES] La mejora genética vegetal tiene como objetivo la obtención de plantas con rasgos mejorados o características novedosas que podrían ayudar a superar los objetivos de sostenibilidad. Para este fin, la biotecnología vegetal necesita incorporar nuevas herramientas de ingeniería genética que combinen una mayor precisión con una mayor capacidad de mejora. Las herramientas de edición genética recientemente descubiertas basadas en la tecnología CRISPR/Cas9 han abierto el camino para modificar los genomas de las plantas con una precisión sin precedentes. Por otro lado, los nuevos enfoques de biología sintética basados en la modularidad y la estandarización de los elementos genéticos han permitido la construcción de dispositivos genéticos cada vez más complejos y refinados aplicados a la mejora genética vegetal. Con el objetivo final de expandir la caja de herramientas biotecnológicas para la mejora vegetal, esta tesis describe el desarrollo y la adaptación de dos nuevas herramientas: una nueva endonucleasa específica de sitio (SSN) y un interruptor genético modular para la regulación de la expresión transgénica. En una primera parte, esta tesis describe la adaptación de CRISPR/Cas12a para la expresión en plantas y compara la eficiencia de las variantes de Acidaminococcus (As) y Lachnospiraceae (Lb) Cas12a con Streptococcus pyogens Cas9 (SpCas9) descritos anteriormente en ocho loci de Nicotiana benthamiana usando expresión transitoria. LbCas12a mostró la actividad de mutagénesis promedio más alta en los loci analizados. Esta actividad también se confirmó en experimentos de transformación estable realizados en tres plantas modelo diferentes, a saber, N. benthamiana, Solanum lycopersicum y Arabidopsis thaliana. Para este último, los efectos mutagénicos colaterales fueron analizados en líneas segregantes sin la endonucleasa Cas12a, mediante secuenciación del genoma descartándose efectos indiscriminados. En conjunto, los resultados muestran que LbCas12a es una alternativa viable a SpCas9 para la edición genética en plantas. En una segunda parte, este trabajo describe un interruptor genético reversible destinado a controlar la expresión génica en plantas con mayor precisión que los sistemas inducibles tradicionales. Este interruptor, basado en el sistema de recombinación del fago PhiC31, fue construido como un dispositivo modular hecho de partes de ADN estándar y diseñado para controlar el estado transcripcional (encendido o apagado) de dos genes de interés mediante la inversión alternativa de un elemento regulador central de ADN. El estado del interruptor puede ser operado externa y reversiblemente por la acción de los actuadores de recombinación y su cinética, memoria y reversibilidad fueron ampliamente caracterizados en experimentos de transformación transitoria y estable en N. benthamiana. En conjunto, esta tesis muestra el diseño y la caracterización funcional de herramientas para la ingeniería del genómica y biología sintética de plantas que ahora ha sido completada con el sistema de edición genética CRISPR/Cas12a y un interruptor genético reversible y biestable basado en el sistema de recombinación del fago PhiC31.
[CAT] La millora genètica vegetal té com a objectiu l'obtenció de plantes amb trets millorats o característiques noves que podrien ajudar a superar els objectius de sostenibilitat. Amb aquesta finalitat, la biotecnologia vegetal necessita incorporar noves eines d'enginyeria genètica que combinen una major precisió amb una major capacitat de millora. Les eines d'edició genètica recentment descobertes basades en la tecnologia CRISPR/Cas9 han obert el camí per modificar els genomes de les plantes amb una precisió sense precedents. D'altra banda, els nous enfocaments de biologia sintètica basats en la modularitat i l'estandardització dels elements genètics han permès la construcció de dispositius genètics cada vegada més complexos i sofisticats aplicats a la millora genètica vegetal. Amb l'objectiu final d'expandir la caixa d'eines biotecnològiques per a la millora vegetal, aquesta tesi descriu el desenvolupament i l'adaptació de dues noves eines: una nova endonucleasa específica de lloc (SSN) i un interruptor genètic modular per a la regulació de l'expressió transgènica . En una primera part, aquesta tesi descriu l'adaptació de CRISPR/Cas12a per a l'expressió en plantes i compara l'eficiència de les variants de Acidaminococcus (As) i Lachnospiraceae (Lb) Cas12a amb la ben establida Streptococcus pyogens Cas9 (SpCas9), en vuit loci de Nicotiana benthamiana usant expressió transitòria. LbCas12a va mostrar l'activitat de mutagènesi mitjana més alta en els loci analitzats. Aquesta activitat també es va confirmar en experiments de transformació estable realitzats en tres plantes model diferents, a saber, N. benthamiana, Solanum lycopersicum i Arabidopsis thaliana. Per a aquest últim, els efectes mutagènics col·laterals van ser analitzats en línies segregants sense l'endonucleasa Cas12a, mitjançant seqüenciació completa del genoma i descartant efectes indiscriminats. En conjunt, els resultats mostren que LbCas12a és una alternativa viable a SpCas9 per a l'edició genètica en plantes. En una segona part, aquest treball descriu un interruptor genètic reversible destinat a controlar l'expressió gènica en plantes amb major precisió que els sistemes induïbles tradicionals. Aquest interruptor, basat en el sistema de recombinació del bacteriòfag PhiC31, va ser construït com un dispositiu modular fet de parts d'ADN estàndard i dissenyat per controlar l'estat transcripcional (encès o apagat) de dos gens d'interès mitjançant la inversió alternativa d'un element regulador central d'ADN. L'estat de l'interruptor pot ser operat externa i reversiblement per acció dels actuadors de recombinació i la seva cinètica, memòria i reversibilitat van ser àmpliament caracteritzats en experiments de transformació transitòria i estable en N. benthamiana. En conjunt, aquesta tesi mostra el disseny i la caracterització funcional d'eines per a l'enginyeria del genòmica i biologia sintètica de plantes que ara ha sigut completat amb el sistema d'edició genètica CRISPR/Cas12a i un interruptor genètic biestable i reversible basat en el sistema de recombinació del bacteriòfag PhiC31.
[EN] Plant breeding aims to provide plants with improved traits or novel features that could help to overcome sustainability goals. To this end, plant biotechnology needs to incorporate new genetic engineering tools that combine increased precision with higher breeding power. The recently discovered genome editing tools based on CRISPR/Cas9 technology have opened the way to modify plant¿s genomes with unprecedented precision. On the other hand, new synthetic biology approaches based on modularity and standardization of genetic elements have enabled the construction of increasingly complex and refined genetic devices applied to plant breeding. With the ultimate goal of expanding the toolbox of plant breeding techniques, this thesis describes the development and adaptation to plant systems of two new breeding tools: a site-specific nuclease (SSNs), and a modular gene switch for the regulation of transgene expression. In a first part, this thesis describes the adoption of the SSN CRISPR/Cas12a for plant expression and compares the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described Streptococcus pyogens Cas9 (SpCas9) in eight Nicotiana benthamiana loci using transient expression experiments. LbCas12a showed highest average mutagenesis activity in the loci assayed. This activity was also confirmed in stable genome editing experiments performed in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, off-target effects in Cas12a-free segregating lines were discarded at genomic level by deep sequencing. Collectively, the results show that LbCas12a is a viable alternative to SpCas9 for plant genome engineering. In a second part, this work describes the engineering of a new reversible genetic switch aimed at controlling gene expression in plants with higher precision than traditional inducible systems. This switch, based on the bacteriophage PhiC31 recombination system, was built as a modular device made of standard DNA parts and designed to control the transcriptional state (on or off) of two genes of interest by alternative inversion of a central DNA regulatory element. The state of the switch can be externally and reversibly operated by the action of the recombination actuators and its kinetics, memory, and reversibility were extensively characterized in N. benthamiana using both transient expression and stable transgenics. Altogether, this thesis shows the design and functional characterization of refined tools for genome engineering and synthetic biology in plants that now has been expanded with the CRISPR/Cas12a gene editing system and the phage PhiC31-based toggle switch.
Bernabé Orts, JM. (2019). Development and characterization of two new tools for plant genetic engineering: A CRISPR/Cas12a-based mutagenesis system and a PhiC31-based gene switch [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/133055
TESIS