Tesi sul tema "Guanosine triphosphatase"

Members of the ubiquitous GTPase superfamily regulate numerous cellular functions. A core group of eight GTPases are present in all domains of life: initiation factor 2, elongation factors Tu and G, protein secretion factors Ffh and FtsY, and the poorly characterized factors YihA, YchF, and HflX. While the first five members have well defined roles in the essential cellular process of protein synthesis, a role for YihA, YchF and HflX in this process has only recently been suggested. Here, a detailed kinetic analysis examining the interaction between HflX and its cellular partners is described. 50S and 70S ribosomal particles function as GTPase activating factors for HflX by stabilizing the nucleotide binding pocket of HflX, inducing a “GTPase activated” state. These data indicates a novel mode of GTPase activation, and suggests a role for HflX in regulating translation.
xii, 185 leaves : ill. (some col.) ; 28 cm

LepA is a highly conserved GTP-binding protein of unknown function. Its amino acid sequence reveals that it is a GTPase with homology to elongation factor G (EF-G). Previous data led to the hypothesis that LepA negatively regulates a posttranslational process such as protein folding. To examine this possibility, two sets of strains carrying mutated alleles encoding molecular chaperones in E. coli were transformed with a lepA expression vector. LepA had a dominant negative effect specifically in a dnaK25 strain whose product exhibits a 20-fold lower ATPase activity compared to wild-type DnaK. The expression of DnaK and other heat-shock proteins is repressed following temperature downshift. Aptly, it was found that temperature shift from 37 degrees Celcius to 15 degrees Celcius in cells harboring a lepA expression vector led to the induction of lepA and downstream lepB. Furthermore, like cold-shock genes, lepA and lepB are induced by sublethal doses of chloramphenicol, although it appears that lep operon induction is related to the antibiotic's action on the 50S ribosome. Due to LepA's insolubility, it could not be confirmed whether it interacts with DnaK, DnaJ or which other proteins it interacts with. Two-dimensional gel electrophoretic analysis revealed the absence of an isoform of OmpA in two lepA deletion strains. It is possible that LepA is involved in a folding pathway that is responsible for the conformation of this isoform. Phylogenetic analysis showed that while LepA is extremely well conserved and has been identified in all completed Bacterial and Eukaryal genomes, it is not present in the completed genomes of any Archaea. Sequence analysis revealed the existence of N-terminus mitochondrial import sequences in Eukaryal LepA orthologues. Additionally, A. thaliana contains a second LepA orthologue that clusters phylogenetically with Synechocystis LepA and has a chloroplastic import sequence. This indicates that plastidal LepA was acquired in A. thaliana (and probably in all plants) through endosymbiosis of an ancestral cyanobacterium. In constrast, mitochondrial LepA are not closely related to those of a- proteobacteria, believed to be the precursors of mitochondria. These findings imply that in sharp contrast to mitochondrial LepA, chloroplastic LepA is under strong evolutionary pressure to remain conserved.

Thesis (Ph. D.)--University of Oregon, 2001.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 90-98). Also available for download via the World Wide Web; free to University of Oregon users.

Protein synthesis is an essential process for all living organisms and is an effective major target for current antibiotics. Elongation factor Tu (EF-Tu) is a highly conserved and essential protein that functions during protein synthesis. EF-Ts interacts with EF-Tu to help maintain a functionally active state of EF-Tu required for cell growth. Although EF-Ts is essential for Escherichia coli, its sequence is poorly conserved. LepA is a highly conserved protein within bacteria and has a similar structure to EF-Tu. In spite of this, LepA has been shown to be non-essential under ideal conditions and the function of LepA still remains elusive. An analysis on the structurally unique aspects of LepA, EF-Tu and EF-Ts was performed here in an effort to gain an understanding on the functions of these proteins. This knowledge, in combination with their unique structural components will provide important tools in developing new and effective antibiotics.
xiii, 177 leaves : col. ill. ; 29 cm

During protein synthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of mRNA-programmed ribosomes in a GTP-dependent manner. To enable future studies on the functional and structural requirement of EF-Tu’s function, a Cysteine-free variant of EF-Tu was constructed suitable for subsequent labelling of the protein and use in kinetic studies. Here, the kinetic properties of three Cysteine-less EF-Tu variants are reported, demonstrating that only the variant with the Alanine substitution in position 81 retains wild-type activity with respect to the interaction with guanine nucleotides, aa-tRNA and the ribosome. To explore a possible tRNA independent pathway for the GTPase activation signal, three residues in domain II of EF-Tu (Arginine 279, Glycine 280, Arginine 283) were mutated; the activity of EF-Tu variants were analyzed. Results suggest that these residues are indeed required for efficient ribosome-dependent stimulation of the GTPase activity of EF-Tu.
x, 85 leaves : ill. (some col.) ; 29 cm

Abstract Ornithine decarboxylase (EC 4.1.1. 17) is the first and the rate-controlling enzyme in polyamine biosynthesis. It decarboxylates L-ornithine to form diamine putrescine. ODC activity in cells is strictly regulated and one of the central elements of ODC regulation is an inhibitory protein called antizyme. Antizyme binds to ODC, inhibits its activity and targets ODC for the proteasomal degradation. Essentiality of polyamines for the normal cell growth and proliferation is well known. Recently their roles in the regulation of several classes of cation channels have been discovered. Some of these channels are expressed abundantly in the brain, which has increased interest in the polyamine metabolism in the central nervous system. In this study guanosine 5'-triphosphate activatable ODC was detected in the rat brain lysates. This activation was more significant after antizyme was separated from ODC. GTP-activatable ODC was more resistant to heat and displayed higher Vmax than kidney ODC. Previously GTP-activatable ODC had been found in mammalian tissues only in some tumors. ODC and antizyme expression in brain was localized by in situ hybridization and immunocytochemistry. Both proteins displayed wide and largely overlapping expression patterns restricted to neurons. The proteins were localized predominantly to cytoplasm at the most brain regions, but antizyme had a main localization in nuclei in some regions of the brain. In addition, the role of one of the most highly conserved regions in eukaryotic ODCs was studied using site-directed mutagenesis. The aspartate-233 to valine mutation was made and detected to increase Km values for the cofactor PLP and the substrate L-ornithine as well as Ki value for the inhibitor DFMO. In another part of this study a transgenic mouse line expressing ODC under the control of viral promotor was generated. The most significant changes in ODC activity were detected in reproductive organs of male mice. The high number of infertile transgenic males supported earlier reports about the importance of balanced polyamine metabolism for spermatogenesis. Infertility of female mice was increased as well, but the involvement of polyamines remained unproven. Transgenic mice were prone to various pathological conditions such as inflammations and tumour formation, which may be due to deregulated polyamine metabolism.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2004.
Includes bibliographical references (leaves 94-103). Also available in electronic version. Access restricted to campus users.

Oxidation of the guanosine (G) moiety, yielding the oxidized lesion 8-hydroxy-2'-deoxyguanosine (oxo82dG), in DNA has become a hallmark biomarker in assessing cellular outcomes induced by oxidative stress. It is well established that the guanosine nucleotide triphosphate pool is also susceptible to oxidative stress and suggested to be more available for oxidation than DNA due to the lack of protective histones and robust repair mechanisms for reducing the levels of all the products of oxidation. The oxidation of guanosine in the nucleotide triphosphate pool, resulting in oxidized guanosine 5'-triphoshate (oxo8GTP), has been overlooked due to the lack of a reliable method. Oxo8GTP has been shown to precede oxidation to G incorporated into DNA and modulate cell processes such as G-protein signaling and RNA synthesis. Evidence is presented in this study of a reliable method to quantify oxo8GTP, a proposed mechanism for the oxidative modification of GTP in the presence of copper and L-ascorbic acid, and evidence of oxo8GTP as an inhibitor of soluble guanylyl cyclase (sGC). A significant induction of oxo8GTP in cell-free preparations as well as in PC12 and HEK 293T cells exposed to physiologically relevant oxidative conditions generated with 10 ?M copper sulphate and 1mM L-Ascorbic Acid (Cu/Asc) is also reported. Exposure to oxidative conditions by Cu/As leads to elevations in oxo8GTP significant enough to result in a reduction of the sGC product, cyclic guanosine monophosphate (cGMP), by as much as half in pure sGC and PC12 cells. GTP is protected from oxidation in the presence of reduced glutathione and this subsequently rescues sGC activity. This suggests that oxo8GTP is produced by free radicals in vivo and can significantly impact neuronal cell functions regulated by sGC activity in the central nervous system such as synaptic plasticity. Alterations in copper homeostasis and oxidative stress have been implicated in several neurodegenerative disorders including Alzheimer's and Parkinson's diseases as well as Amyotrophic Lateral Sclerosis. Based upon evaluation of the data presented herein, we hypothesize that neuronal deficiencies in such disorders might be due to oxidation of the GTP pool and the ensuing effects on neuronal function.

A synthetic pathway towards the cap-structure of 2,2,7-trimethylguanosine containing a methylene modified triphosphate bridge have been investigated. The modification to the triphosphate bridge is hoped to slow down cap degradation and give the connected  oligunucleotide an increased lifetime. This could result in an better understanding of nuclear transport of oligonucleotides and could thereby helping to develop new treatments for different diseases. The synthesis relies on a coupling reaction between the 2,2,7-trimethylguanosine 5’phosphate and 2’-O-methyladenosine with a 5’-pyrophosphate where the central oxygen has been replaced by a methylene group. The reaction pathway consists of 9 steps of which 8 steps have been successfully performed. The last step, which includes a coupling reaction, was attempted but without successful identification and isolation of the cap-structure, and will need further attention. The reaction has been performed in a milligram scale with various yields.

Presentation utförd

Malaria is a fatal tropical disease affecting billions of people in impoverished countries world-wide. An alarming fact is that a child in Africa dies of malaria every 30 seconds that amounts to 2500 children per day (www.who.int/features/factfiles). Malaria is caused by the intraerythrocytic forms of Plasmodium species, notably P. falciparum, P. vivax, P. ovale and P. malariae (Hyde 2007). The spread of drug-resistant strains, failure of vector control programs, rapid growth rate of the parasite, and lack of a vaccine have further exacerbated the effects of malaria on economic development and human health. It is therefore imperative that novel drug targets are developed or current antimalarial drugs optimized (Foley and Tilley 1998). One such target is folate biosynthesis, given that folates and their derivatives are required for the survival of organisms (Muller et al. 2009). DHFR and DHPS are currently the only folate targets exploited however, their antifolate drugs are almost useless against parasite resistant strains. As such, guanosine-5’triphosphate cyclohydrolase I (GTPCHl) among other antifolate candidates are considered for intervention (Lee et al. 2001). Knock-out studies (of P. falciparum gtpchI) resulted in the suppression of DHPS activity (Nzila et al. 2005). Additionally, gtpchI amplified 11-fold in P. falciparum strains resistant to antifolates due to mutations in dhps and dhfr and this may be a mechanism for the compensation of reduced flux of folate intermediates (Kidgell et al. 2006; Nair et al. 2008). Over-expression of P. falciparum proteins in E. coli remains a challenge mainly due to the A+T rich Plasmodium genome resulting in a codon bias. This results in the expression of recombinant proteins as insoluble proteins sequestered in inclusion bodies (Carrio and Villaverde 2002; Mehlin et al. 2006; Birkholtz et al. 2008a). Comparative expression studies were conducted of native GTPCHI (nGTPCHI), codon optimized GTPCHI (oGTPCHI) and codon harmonized (hGTPCHI) in various E. coli cell lines, using alternative media compositions and co-expression with Pfhsp70. The nGTPCHI protein did not express because the gene consisted of codons rarely used by E. coli (codon bias). The expression levels of purified hGTPCHI were a greater in comparison to oGTPCHI using the different expression conditions. This is because codon-harmonization involves substituting codons to replicate the codon frequency preference of the target gene in P. falciparum, as such the translation machinery matches that of Plasmodium (Angov et al. 2008). Furthermore, greater expression levels of GTPCHI were achieved in the absence of Pfhsp70 due to expression of a possible Nterminal deletion product or E. coli protein. Purification conditions could be improved to obtain homogenous GTPCHI and further analysis (mass spectrometry and enzyme activity assays) would be required to determine the nature of soluble GTPCHI obtained. To improve the expression of soluble proteins the wheat germ expression system was used as an alternate host. However, GTPCHI expression was not effective, possibly due to degradation of mRNA template or the absence of translation enhancer elements.
Dissertation (MSc)--University of Pretoria, 2011.
Biochemistry
unrestricted

A synthetic pathway towards the cap-structure of 2,2,7-trimethylguanosine containing a methylene modified triphosphate bridge have been investigated. The modification to the triphosphate bridge is hoped to slow down cap degradation and give the connected  oligunucleotide an increased lifetime. This could result in an better understanding of nuclear transport of oligonucleotides and could thereby helping to develop new treatments for different diseases. The synthesis relies on a coupling reaction between the 2,2,7-trimethylguanosine 5’phosphate and 2’-O-methyladenosine with a 5’-pyrophosphate where the central oxygen has been replaced by a methylene group. The reaction pathway consists of 9 steps of which 8 steps have been successfully performed. The last step, which includes a coupling reaction, was attempted but without successful identification and isolation of the cap-structure, and will need further attention. The reaction has been performed in a milligram scale with various yields.
Presentation utförd

La preorganització electrostàtica del centre actiu s'ha postulat com el mecanisme genèric de l'acció dels enzims. Així, alguns residus "estratègics" es disposarien per catalitzar reaccions interaccionant en una forma més forta amb l'estat de transició, baixant d'aquesta manera el valor de l'energia dactivació g cat. S'ha proposat que aquesta preorientació electrostática s'hauria de poder mostrar analitzant l'estabilitat electrostàtica de residus individuals en el centre actiu.
Ras es una proteïna essencial de senyalització i actúa com un interruptor cel.lular. Les característiques estructurals de Ras en el seu estat actiu (ON) són diferents de les que té a l'estat inactiu (OFF). En aquesta tesi es duu a terme una anàlisi exhaustiva de l'estabilitat dels residus del centre actiu deRas en l'estat actiu i inactiu.
The electrostatic preorganization of the active site has been put forward as the general framework of action of enzymes. Thus, enzymes would position "strategic" residues in such a way to be prepared to catalyze reactions by
interacting in a stronger way with the transition state, in this way decreasing the activation energy g cat for the catalytic process. It has been proposed that
such electrostatic preorientation should be shown by analyzing the electrostatic stability of individual residues in the active site.
Ras protein is an essential signaling molecule and functions as a switch in the
cell. The structural features of the Ras protein in its active state (ON state) are different than those in its inactive state (OFF state). In this thesis, an exhaustive analysis of the stability of residues in the active and inactive Ras active site is performed.

Guanosine triphosphatases (GTPases) are signaling mediators involved in regulation of diverse cellular processes including regulation of the actin cytoskeleton, gene transcription, cell cycle regulation, apoptosis and transformation. Regulatory, proteins including G protein coupled receptors (GPCR), GTPase activating proteins (GAP) and guanine nucleotide exchange factors (GEF) influence the activity of GTPases. Balanced regulation of GTPase activity is critical in coordinating normal cellular responses. This thesis addresses the contributions of GTPase regulators in cellular growth control, differentiation and transformation. Over-expression of G2A or PAR-1, two GPCRs, in NIH 3T3 fibroblasts induced a full range of phenotypes characteristic of oncogenic transformation. Co-expression of dominant negative Rho or LscRGS (Lbc's second cousin regulator of G protein signaling domain), a negative regulator of Gα₁₂ and Gα₁₃ GTPases, suppressed transformation via these GPCRs. Activation of Gα₁₂, Gα₁₃ and Rho GTPases are thus required for transformation via these GPCRs. Gα₁₂ and Gα₁₃ are unique in that they are activated upstream of Rho. Moreover, Rho GTPase activity is regulated via GEFs. Gct-mediated activation of GEFs and downstream smaller molecular weight GTPases appears to be an important mechanism of linking divergent GTPases downstream of GPCR activation. To elucidate the role of Gα₁₂ and Gα₁₃ GTPases in lymphocyte development, transgenic mice expressing LscRGS were generated. Analyses of lymphocytes from these mice revealed that LscRGS expression did not overtly affect lymphocyte development. These results indicate that Gα₁₂ and Gα₁₃ are not required for lymphocyte development. Rho and Cdc42 are two GTPases involved in lymphocyte development. Previous studies by others demonstrated that loss of Rho function partially blocked differentiation and survival of CD4/CD8- double negative (DN) thymocytes and expression of another Rho family GTPase, Cdc42, enhanced the proliferative capacity of DN thymocytes. In addition, results from other studies revealed that expression of activated Rho augments positive selection and induces CD4⁺/CD8- and CD4/CD8⁺ single positive (SP) thymocyte hypersensitivity to TCR-induced proliferation in vitro. Dbs is a Rho- and Cdc42-activating GEF normally expressed in thymus. To determine how Dbs influences lymphocyte development, transgenic mice were generated expressing an activated form of Dbs. Expression of activated Dbs in lymphocytes promoted the accumulation of early thymocytes and restricted the production of mature thymocytes. Activated Dbs expression also led to increased proliferation of DN thymocytes. The Dbs transgene caused reduced numbers of SP thymocytes and mature splenic T lymphocytes. In addition, transgenic CD4⁺/CD8⁺ double positive (DP) thymocytes expressed higher levels of T cell receptor (TCR) and were hypersensitive to apoptosis induced by injection of anti-CD3. Moreover, Dbs transgenic thymocytes displayed impaired positive selection. Thymocyte culture experiments revealed that proliferation in response to anti-CD3 was reduced in SP thymocytes from Dbs transgenic mice. Expression of activated Dbs, a positive regulator of Rho and Cdc42, promoted the accumulation of DN thymocytes; this is the opposite of the DN phenotype observed in thymocytes lacking Rho function and similar to the phenotype displayed by Cdc42 transgenic mice. Thus the accumulation of DN thymocytes is likely to occur via Rho and/or Cdc42 activation. Activated Dbs expression also caused reduced in vitro SP thymocyte proliferation in response to TCR cross-linking and impaired thymocyte positive selection. These results are contrary to previous reports describing transgenic mice expressing activated Rho; thus, impaired thymocyte proliferation and positive selection in Dbs transgenic mice is likely to involve a pathway independent of Rho. Results presented in this thesis provide insights into the contributions of GTPase regulators in regulation of cellular growth control, differentiation and transformation.

蛋白質合成的延伸階段由兩個延伸因子推動，而這兩個延伸因子與核糖體的結合點同樣位於核糖體柄的底部。作為GTP酶，這兩個延伸因子本身無活性，需要依賴GTP酶相關中心在適當的時候把他們轉化為活性酶。真核生物的GTP酶相關中心由28S核糖體核糖核酸58個鹼基、P0（P1/P2）₂五聚體蛋白複合體及柄基蛋白eL12組成。由於核糖體柄的動態結構，這個區域在現今的真核生物核糖體結構研究中仍然未能解構，而我們的研究成功判斷出核糖體柄複合結構的特徵。我們確定了穩定P1/P2異源二聚體的相互作用，指出P1/P2異源二聚體比P2同源二聚體擁有較高的構象穩定性。同時我們發現了P1第三螺旋上一個外露的疏水區，對於P1/P2異源二聚體與P0的結合有重要的作用。就此我們決定了P0的兩個脊柱螺旋為P1/P2異源二聚體的結合點。利用同源模擬法及蛋白突變，我們提出了有關核糖體柄結構的新模型。在這個模型中，結合於P0上的兩個異源二聚體以P2/P1：P1/P2序列。我們提出的模型能夠解釋每個P-蛋白對GTP酶活性的不同貢獻，以及P0上兩個P1/P2異源二聚體的功能協同性。這個模型中核糖體柄結構的方向性，最能配合核糖體柄募集延伸因子的功能。基於對核糖體柄的研究，我們進一步研究柄基蛋白eL12並提出初步數據顯示eL12與核糖體柄之間的直接互動。這個研究結果提出，eL12的功能很可能是透過與核糖體柄的直接活動來傳遞結合及激活訊號。我們就GTP酶相關中心的研究補充了對真核生物核糖體結構的研究，加深了對GTP酶相關中心如何推動蛋白質合成的理解。
The elongation cycle of protein synthesis is driven by two elongation factors that bind to overlapping sites at the base of the ribosomal stalk. Both factors have limited inherent GTPase activity and they rely on the GTPase associated centre to activate GTP hydrolysis at appropriate times during elongation. In eukaryotes, this region consists of a 58-base 28S ribosomal RNA, the P0(P1/P2)₂ pentameric stalk complex and the stalk base protein eL12. Due to the dynamic nature of the ribosomal stalk, this region remains as a missing piece in the high-resolution structural studies of the eukaryotic ribosome. In this work, we have characterized the structural organization of the stalk complex. We have identified the stabilizing interactions within P1/P2 heterodimer and showed that P1/P2 heterodimer is preferred over P2 homodimer due to its higher conformational stability. We have also identified an exposed hydrophobic patch on helix-3 of P1 that is important for anchoring P1/P2 heterodimers to P0 and we havemapped two spine helices on P0 as the binding sites for P1/P2 heteodimer. Based on homology modelling and mutagenesis experiments, we have proposed a new model of the eukaryotic stalk complex where the two heterodimers display a P2/P1:P1/P2 topology on P0. Our model provides an explanation for the difference of GTPase activities contributed by each P-protein and the functional contribution of the hydrophobic loop between the two spine helices of P0. Our model represented the stalk complex in an orientation that is the most effective for recruiting translation factors to their binding sites. As an extension to our studies, we have preliminary data showing direct interaction between eL12 and stalk complex. This is a strong suggestion that eL12 contributes to its functional role by transmitting signal for factor binding and activation through direct interaction with the stalk complex. Our work on the GTPase associated centre has supplemented the structural studies of the eukaryotic ribosome and provided a betterpicture of how the GTPase associated centre contributes to the high efficiency of protein synthesis.
Detailed summary in vernacular field only.
Yu, Wing Heng Conny.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 117-125).
Abstract also in Chinese.
Chapter i. --- Abstract --- p.1
Chapter ii. --- 摘要 --- p.3
Chapter iii. --- Acknowledgments --- p.4
Chapter iv. --- Disclaimer --- p.5
Chapter v. --- List of figures --- p.6
Chapter vi. --- Table of Contents --- p.7
Chapter Chapter 1. --- Project Background and Objectives --- p.10
Chapter 1.1. --- The ribosome --- p.10
Chapter 1.1.1. --- Its components: ribosomal RNA and proteins --- p.10
Chapter 1.1.2. --- Its function: protein translation --- p.12
Chapter 1.2. --- The GTPase Associated Centre --- p.13
Chapter 1.2.1. --- P-complex: P0, P1 and P2 --- p.14
Chapter 1.2.2. --- Stalk base protein: eL12 --- p.16
Chapter 1.3. --- Project objectives --- p.17
Chapter 1.3.1. --- Structural organization of the P-complex --- p.18
Chapter 1.3.2. --- Characterization of the interaction between eL12 and P-complex --- p.19
Chapter Chapter 2. --- Methods and Materials --- p.20
Chapter 2.1. --- DNA Techniques --- p.20
Chapter 2.1.1. --- Agarose gel electrophoresis of DNA --- p.20
Chapter 2.1.2. --- Sub-cloning --- p.21
Chapter 2.1.3. --- Site-directed mutagenesis --- p.23
Chapter 2.2. --- RNA Techniques --- p.24
Chapter 2.2.1. --- in vitro transcription and purification of RNA --- p.24
Chapter 2.2.2. --- Agarose gel electrophoresis of RNA --- p.25
Chapter 2.2.3. --- Electrophoretic mobility shift assay (EMSA) --- p.26
Chapter 2.3. --- General protein techniques --- p.27
Chapter 2.3.1. --- Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.27
Chapter 2.3.2. --- Native PAGE for acidic proteins --- p.28
Chapter 2.3.3. --- Protein transfer and Western blotting --- p.29
Chapter 2.4. --- Expression and purification of recombinant proteins --- p.30
Chapter 2.4.1. --- Preparation of E. coli competent cells --- p.30
Chapter 2.4.2. --- Transformation and bacterial culture --- p.31
Chapter 2.4.3. --- Protein extraction by cell lysis. --- p.32
Chapter 2.4.4. --- Purification of P2 and mutants --- p.33
Chapter 2.4.5. --- Purification of P1 and mutants --- p.36
Chapter 2.4.6. --- Purification of His-P0 and mutants --- p.39
Chapter 2.4.7. --- Purification of P1/P2 heterodimer and mutants --- p.41
Chapter 2.4.8. --- Reconstitution and purification of P0(P1/P2)₂ complex and mutants --- p.43
Chapter 2.4.9. --- Purification of eL12 and mutants --- p.44
Chapter 2.5. --- Preparation of rat Elongation factor 2 (EF-2) --- p.46
Chapter 2.5.1. --- Preparation of liver lysate --- p.46
Chapter 2.5.2. --- Purification of rat EF-2 --- p.47
Chapter 2.6. --- Circular dichroism (CD) spectrometry --- p.49
Chapter 2.6.1. --- Chemical denaturation --- p.50
Chapter 2.6.2. --- Thermal denaturation --- p.51
Chapter 2.7. --- Limited proteolysis --- p.52
Chapter 2.8. --- Light scattering (LS) experiments --- p.53
Chapter 2.8.1. --- Size exclusion chromatography coupled with light scattering detection (SEC/LS) --- p.53
Chapter 2.8.2. --- Dynamic light scattering (DLS) --- p.54
Chapter 2.9. --- in vitro binding assay using NHS-activated Sepharose --- p.55
Chapter 2.10. --- Homology modelling --- p.56
Chapter 2.10.1. --- Sequence alignment --- p.56
Chapter 2.10.2. --- Modelling using UCSF Chimera built-in Modeller --- p.57
Chapter 2.10.3. --- Modelling using Modeller scripts --- p.58
Chapter 2.11. --- Buffers and reagents --- p.60
Chapter 2.11.1. --- Media for general bacterial culture --- p.60
Chapter 2.11.2. --- Reagents for DNA and RNA gel electrophoresis --- p.61
Chapter 2.11.3. --- Reagents for SDS-PAGE and native PAGE --- p.62
Chapter 2.11.4. --- Reagents for Western blotting --- p.62
Chapter 2.12. --- Sequences of DNA oligos --- p.64
Chapter 2.12.1. --- Primers for P1 mutants --- p.64
Chapter 2.12.2. --- Primers for P0 mutants --- p.65
Chapter 2.12.3. --- Primers for eL12 and its mutants --- p.66
Chapter 2.12.4. --- DNA template for in vitro transcription --- p.68
Chapter Chapter 3. --- Structural Organization of the Eukaryotic Stalk Complex --- p.69
Chapter 3.1. --- Introduction --- p.69
Chapter 3.2. --- Results --- p.71
Chapter 3.2.1. --- Homology modelling of P1/P2 heterodimer --- p.71
Chapter 3.2.2. --- P1/P2 heterodimer is stabilized by a hydrophobic interface --- p.74
Chapter 3.2.3. --- Helix-3 of P1 plays a vital role in P-complex formation --- p.78
Chapter 3.2.4. --- C-terminal tails are not involved in P-complex formation --- p.80
Chapter 3.2.5. --- Spine helices of P0 are the binding sites for P1/P2 heterodimers --- p.83
Chapter 3.2.6. --- Homology modelling of the pentameric complex --- p.86
Chapter 3.3. --- Discussion --- p.89
Chapter 3.3.1. --- Comparison between homology model and structure of P1/P2 heterodimer --- p.89
Chapter 3.3.2. --- Biological significance of P2/P1:P1/P2 topology --- p.92
Chapter 3.4. --- Towards structure determination of P-complex --- p.97
Chapter Chapter 4. --- Characterization of the interaction between eL12 and P-complex. --- p.99
Chapter 4.1. --- Introduction --- p.99
Chapter 4.2. --- Results --- p.100
Chapter 4.2.1. --- Homology modelling of human eL12 --- p.100
Chapter 4.2.2. --- Characterization of recombinant eL12 --- p.103
Chapter 4.2.3. --- eL12 directly interacts with P-complex via its N-terminal residues --- p.106
Chapter 4.3. --- Discussion --- p.108
Chapter 4.4. --- Towards structure determination of eL12 --- p.111
Chapter Chapter 5. --- Conclusion and future work --- p.114
Chapter 5.1. --- Proposed working mechanism of eukaryotic GTPase Associated Centre --- p.114
Chapter 5.1.1. --- Anchorage to the ribosome through RNA binding --- p.114
Chapter 5.1.2. --- P1/P2 heterodimers are bound to P0 in a P2/P1:P1/P2 topology --- p.114
Chapter 5.1.3. --- eL12 as functional player in the GTPase associated centre. --- p.115
Chapter 5.2. --- Future work --- p.116
Chapter vii. --- References --- p.117

ROP GTPases are crucial regulators of pollen tube growth. The Rop GTPase family in maize consists of nine known rop genes, ropl-rop9. A subset of these genes (rop2, rop8, and rop9) are expressed in pollen. The rop2 and rop9 genes are a highly conserved duplicate gene pair of ancient origin. The rop2/rop9 duplicate gene pair displays differential expression in mature and germinated pollen, suggesting different roles for the genes in the process of male gametophyte development. To explore ROP2 function in maize, five Mutator transposon insertions in the rop2 gene were isolated (rop2::Mu alleles). I showed that three of the rop2::Mu alleles displayed reduced transmission through the male and were associated with reduced levels of ROP2-mRNA. Interestingly, the rop2::Mu male-specific transmission defect was apparent only when wild-type pollen was also present, an indication that the mutation reduces the competitive ability of the rop2 gametophytes. Dual pollination and pollen mixing experiments indicated that this competitive disadvantage is expressed by the majority of the mutant gametophytes, and that expression of the phenotype is associated with a delay in the ability to accomplish fertilization. Using the waxy phenotypic marker (linked to rop2 via a reciprocal translocation) to distinguish between rop2::Mu and wild-type pollen derived from heterozygous plants, I demonstrated that the delay is associated with a defect in early progamic development (i.e, germination and early pollen tube growth). The defect was detectable in vivo as early as 15 minutes after pollination. However, quantitative measurements provided no indication that the rop2 mutation affects pollen tube growth in the style. Finally, investigations focusing on the final stages of pollen function raise the possibility that a defect in the very last stages (i.e. either pollen tube guidance through the micropyle to the egg sac, or fertilization of the egg sac) may also contribute to the rop2 mutant delay. This work provides direct in vivo evidence confirming a role for Rop in male gametophyte development, and is the first study to demonstrate a role for Rop in the early stages of post-pollination gametophytic function.
Graduation date: 2005

Thesis (Ph.D.)--Indiana University, 2006.
Title from screen (viewed on Apr. 27, 2007) Department of Pharmacology & Toxicology, Indiana University-Purdue University Indianapolis (IUPUI) Includes vita. Includes bibliographical references (leaves 181-239)

Xue, Yan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 95-102).
Abstract also in Chinese.
Thesis committe --- p.2
Statement --- p.3
Abstract --- p.4
Acknowledgement --- p.8
General abbreviations --- p.10
Abbreviations of chemicals --- p.13
List of figures --- p.15
List of tables --- p.16
Table of contents --- p.17
Chapter Chapter 1 --- General Introduction
Chapter 1.1 --- Impact of bacterial blight on rice production --- p.25
Chapter 1.2 --- The plant immune system --- p.25
Chapter 1.2.1 --- Preformed resistance --- p.25
Chapter 1.2.2 --- PAMP triggered immunity (PTI) --- p.26
Chapter 1.2.3 --- Effecter triggered immunity (ETI) --- p.27
Chapter 1.2.3.1 --- R genes --- p.27
Chapter 1.2.3.2 --- Hypersensitive responses (HR) --- p.27
Chapter 1.2.3.3 --- Systemic acquired resistance (SAR) --- p.28
Chapter 1.2.3.3.1 --- Salicylic acid is required for SAR establishment --- p.28
Chapter 1.2.3.3.2 --- Involvement of lipid-based molecules in SAR signaling --- p.28
Chapter 1.2.3.3.3 --- NPR1: the master regulator of SAR --- p.29
Chapter 1.2.3.3.4 --- Expression of pathogenesis related (PR) genes --- p.29
Chapter 1.2.4 --- Interaction between SA and JA --- p.29
Chapter 1.2.5 --- Other important signaling components in plant defense responses --- p.30
Chapter 1.2.5.1 --- G proteins --- p.30
Chapter 1.2.5.2 --- G proteins in defense responses --- p.30
Chapter 1.3 --- OsGAPl is a C2 (protein kinase C conserved region 2) domain harboring GTPase activating protein --- p.32
Chapter 1.4 --- OsYchFl is a GTPase and an interacting partner of OsGAPl --- p.32
Chapter 1.5 --- Hypothesis and objectives of this research --- p.33
Chapter Chapter 2 --- materials and methods
Chapter 2.1 --- Materials --- p.35
Chapter 2.1.1 --- Chemicals and reagents --- p.39
Chapter 2.1.2 --- Commercial kits --- p.40
Chapter 2.1.3 --- Primers used --- p.41
Chapter 2.1.4 --- Equipment and facilities used: --- p.47
Chapter 2.1.5 --- "Buffer, solution, gel and medium:" --- p.47
Chapter 2.2 --- Methods: --- p.51
Chapter 2.2.1 --- Culture of bacterial strains --- p.51
Chapter 2.2.2 --- Composition of medium used in this work for cultivating bacterial strains: --- p.51
Chapter 2.2.3 --- Plant growth and treatment --- p.52
Chapter 2.2.3.1 --- Surface sterilization of Arabidopsis thaliana seeds --- p.52
Chapter 2.2.3.2 --- Seed germination and Arabidopsis plant growth --- p.52
Chapter 2.2.4 --- Generation of transgenic Arabidopsis --- p.53
Chapter 2.2.4.1 --- Agrobacterium-mediated Arabidopsis transformation --- p.53
Chapter 2.2.5 --- Pathogen inoculation test --- p.54
Chapter 2.2.6 --- Molecular cloning --- p.54
Chapter 2.2.6.1 --- DNA sequencing: --- p.55
Chapter 2.2.6.2 --- Transformation of E. coli strains: --- p.55
Chapter 2.2.6.3 --- Transformation of Agrobacteria by electroporation --- p.55
Chapter 2.2.7 --- DNA and RNA extraction --- p.56
Chapter 2.2.7.1 --- Plasmid DNA extraction from bacterial cells --- p.56
Chapter 2.2.7.2 --- Genomic DNA extraction from plant tissues --- p.56
Chapter 2.2.7.3 --- RNA extraction from plant tissues --- p.56
Chapter 2.2.8 --- Northern blot --- p.57
Chapter 2.2.9 --- Subcellular localization studies --- p.58
Chapter 2.2.9.1 --- Transformation of tobacco BY-2 cells --- p.58
Chapter 2.2.9.2 --- Maintenance of transgenic tobacco BY-2 cells --- p.59
Chapter 2.2.9.3 --- Confocal microscopy --- p.59
Chapter 2.2.9.4 --- Electron microscopy --- p.59
Chapter 2.2.10 --- Bimolecular fluorescence complementation studies (BiFC) --- p.60
Chapter 2.2.10.1 --- Construct making --- p.61
Chapter 2.2.10.2 --- Preparation of rice protoplasts --- p.61
Chapter 2.2.10.3 --- PEG-mediated transfection --- p.62
Chapter 2.2.10.4 --- Detection of protein-protein interaction --- p.62
Chapter Chapter 3 --- Results
Chapter 3.1 --- OsGAPl interacts with OsYchFl in vivo --- p.63
Chapter 3.1.1 --- Construction of vectors for BiFC transient assay in rice protoplasts --- p.64
Chapter 3.1.2 --- BiFC assay in rice protoplasts revealed in vivo interaction between the OsGAPl and the OsYchFl proteins --- p.66
Chapter 3.2.1 --- Subcellular localization of OsGAPl --- p.68
Chapter 3.2.2 --- Localization of OsGAPl and OsYchFl in rice leaves revealed by electron microscopy --- p.70
Chapter 3.3 --- Functional characterization of OsYchFl
Chapter 3.3.1 --- Characterization of Arabidopsis YchF1 knockdown mutant --- p.75
Chapter 3.3.2 --- Complementation of AtYchF1 knockdown Arabidopsis --- p.77
Chapter 3.3.3.1 --- Pathogen inoculation test --- p.80
Chapter Chapter 4 --- Discussion
Chapter 4.1 --- Significance of the project --- p.85
Chapter 4.2 --- In vivo interaction between OsGAPl and OsYchFl --- p.86
Chapter 4.3 --- OsGAPl is located either inside the cytosol or on the plasma membrane in transgenic tobacco BY-2 cells --- p.87
Chapter 4.4 --- Study of wounding effect on the subcellular localization of OsGAPl and OsYchFl at whole plant level by EM --- p.88
Chapter 4.5 --- OsYchFl functions as a negative regulator of defense responses in A.thaliana --- p.90
Chapter 4.6 --- Conclusion --- p.92
References --- p.95
Appendix --- p.103

Intracellular dNTP pool sizes are highly asymmetric, with dGTP usually comprising 5 to 10% of the sum of the dNTP pools. The work presented in this dissertation addresses the question of whether the underrepresentation of dGTP is related to its potential to be oxidized by reactive oxygen species. 8-oxo-guanine is important in oxidative mutagenesis, and current evidence indicates that this lesion arises in DNA partly through oxidation of dGTP, followed by incorporation of 8-oxo-dGTP into DNA. The bacterial MutT protein and its mammalian homolog catalyze the hydrolysis of 8-oxo-dGTP to 8-oxo-dGMP in vitro. It is a widely accepted premise that the primary function of these enzymes is to remove 8-oxo-dGTP from the nucleotide pool of cells so that it cannot be used as a substrate for DNA synthesis. However, this model has been called into question by observations that some mutT strains of E. coli display a mutator phenotype when grown anaerobically, and by kinetic studies that showed 8-oxo-dGTP to be a poor DNA polymerase substrate. In this study, the dNTP pools of mammalian cells cultured in varying oxygen conditions were measured, with the expectation that the dGTP pool would expand under low oxygen conditions if it were a target for damage by reactive oxygen species. HeLa cells cultured in 2% 0��� showed no change in the dGTP pool when compared to cells cultured in 20% 0���; however, in V79 cells, the dGTP pool did expand in 2% 0���. This result was not specific to the dGTP pool, as pools of dATP and dTTP also increased when V79 cells were cultured at 2% 0���. These results suggest that there may be increased turnover of the dGTP pool when cells are cultured in high oxygen, but these experiments did not address the reason for this oxygen-dependent change. In order to determine whether 8-oxo-dGTP accumulates to levels that are sufficient to cause mutagenesis in cells, an analytical method for the measurement of 8-oxo-dGTP from cell extracts was developed. By use of this method, which involves reversed-phase high performance liquid chromatography coupled with electrochemical detection, no 8-oxo-dGTP was detected in mutT E. coli cells, even when they were cultured in the presence of H���0���. The estimated upper limit of 8-oxo-dGTP in these cells is about 240 molecules per cell, which corresponds to an intracellular concentration of approximately 0.34 ��M. When 8-oxo-dGTP was added at this concentration to an in vitro DNA replication system in which replication errors could be scored as mutations, along with the four normal dNTPs at their estimated intracellular concentrations, there was no detectable effect on the frequency of mutation. Therefore, the presence of 8-oxo-dGTP at physiologically relevant concentrations does not appear to be significantly mutagenic. The results presented in this dissertation suggest that the mechanism by which the MutT enzyme counteracts mutagenesis should be reevaluated.
Graduation date: 2003

Indiana University-Purdue University Indianapolis (IUPUI)
The immune system initiates tissue repair following injury. In response to sterile tissue injury, neutrophils infiltrate the tissue to remove tissue debris and subsequently undergo apoptosis. Proper clearance of apoptotic neutrophils in the tissue by recruited macrophages, in a process termed efferocytosis, is critical to facilitate the resolution of inflammation and tissue repair. However, the events leading to suppression of sterile inflammation following efferocytosis, and the contribution of other innate cell types are not clearly defined in an in vivo setting. Using a sterile mouse peritonitis model, we identified IL-4 production from efferocytosing macrophages in the peritoneum that activate invariant NKT cells to produce cytokines including IL-4 and IL-13. Importantly, IL-4 from macrophages functions in autocrine and paracrine circuits to promote alternative activation of peritoneal exudate macrophages and augment type-2 cytokine production from NKT cells to suppress inflammation. The increased peritonitis in mice deficient in IL-4, NKT cells, or IL-4Ra expression on myeloid cells suggested that each is a key component for resolution of sterile inflammation. The phagocyte NADPH oxidase, a multi-subunit enzyme complex we demonstrated to require a physical interaction between the Rac GTPase and the oxidase subunit gp91phox for generation of reactive oxygen species (ROS), is required for production of ROS within macrophage phagosomes containing ingested apoptotic cells. In mice with X-linked chronic granulomatous disease (X-CGD) that lack gp91phox, efferocytosing macrophages were unable to produce ROS and were defective in activating iNKT during sterile peritonitis, resulting in enhanced and prolonged inflammation. Thus, efferocytosis-induced IL-4 production and activation of IL-4-producing iNKT cells by macrophages are immunomodulatory events in an innate immune circuit required to resolve sterile inflammation and promote tissue repair.