Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-23Bitstream added on 2014-06-13T19:01:10Z : No. of bitstreams: 1 antonio_t_dr_araiq.pdf: 685450 bytes, checksum: 38953886194d585fde26a8d2130709e0 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Mutantes foram obtidos através de tratamento mutagênico dos esporos de S. clavuligerus ATCC 27064 com metil-metanosulfonato. Um total de 822 colônias, obtidas por repiques sucessivos, foram selecionadas em testes qualitativos e/ou quantitativos. O melhor mutante S. clavuligerus 45.41, mostrou-se de duas a quatro vezes mais produtivo que a linhagem selvagem, e manteve-se estável após 35 repiques sucessivos ao longo deste estudo. O comportamento das linhagens foi investigado pela adição de diferentes diaminas ao meio de cultivo, uma a uma, na ausência de lisina. Também se investigou duas fontes distintas de C, além de compostos como o ácido 2,6- diaminopimélico. Um planejamento de dois fatores (cadaverina e lisina), três níveis, baseado no ponto central, faces centradas, indicou que a adição de lisina aumenta a produção de CefC para ambas as linhagens. O efeito positivo da cadaverina foi observado principalmente na linhagem mutante 45.41. Resultados obtidos em processo de batelada, em biorreator de bancada confirmaram as diferenças observadas entre as linhagens nos cultivos em frascos agitados. Procedimento de mutagênese clássica foi utilizado em conjunto com critérios bem definidos para se estabelecer um meio de cultura apropriado, com o objetivo de alcançar um aumento significativo da produção de CefC. Este é o primeiro estudo utilizando um mutante de S. clavuligerus obtido por mutagênese clássica, cuja produção de CefC é de três vezes maior que a da linhagem selvagem, em meios contendo diaminas / Mutants were obtained by treating S. clavuligerus ATCC 27064 spores with methyl-methanesulfonate. A total of 822 colonies, obtained by successive sampling, were selected by qualitative and/or quantitative tests. The best mutant, S. clavuligerus 45.41, was two to four times more productive than the wild-type strain, and remained stable even after 35 successive samplings throughout the study. Strains behavior was investigated by adding different diamines in media, one by one, in the absence of lysine. Also investigated two different sources of C, and compounds such as 2,6-diaminopimelic acid. The two-factor (cadaverine and lysine), three-level, central composite-based, face-centered experimental design indicates that adding lysine increases CephC production for both strains alike. The positive effect of cadaverine was observed mainly in the Mutant 45.41 strain. Results obtained in batch-processes in a bench-scale bioreactor confirmed differences among strains observed in shaken flasks cultures. Classical mutagenesis procedures in conjunction with the adoption of well-defined criteria to establish an appropriate culture medium promoted a significant improvement in CephC production. It is the first study indicating an increase in CephC production in media containing diamines employing a mutant of S. clavuligerus obtained by classical mutagenesis

Student Number : 0201444H - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Due to the paucity of sensitive mutation markers available for studying mycobacterial species it was decided to explore the suitability of mycobacteriophage L5 as an analogous mutation detection system to phage Lambda in E. coli. The system relies on the detection of an increased production of clear plaque mutants (CPM) arising from turbid plaques, in response to DNA damage. A number of L5 phage experimental tools were developed and optimized, including a lysogen-based CPM confirmation assay. The mutant induction system was applied to wild type M. smegmatis mc2155 and its recA mutant, dinP mutant as well as an M. smegmatis(L5) lysogen. The lysogen system proved to be insensitive with respect to mutant induction since elevated CPM frequencies could not be detected. Interestingly, the wild type M. smegmatis mc2155 system demonstrated slightly elevated CPM frequencies in response to transfection of untreated L5 on UV irradiated host cells. This result suggests that a host SOS mutagenic system is able to act on normal, undamaged DNA bases. The involvement of the SOS response in untargeted mutagenesis was confirmed by the abrogation of increased CPM frequency, in an M. smegmatis recA mutant. This data supports suggestions that RecA is responsible for the control of the SOS response. The M. smegmatis dinP mutant system showed a decrease in CPM frequency which supports evidence that this gene does have mutator polymerase activity, as is in seen E. coli dinP homologues.

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.

NADH:Cytochrome b5 Reductase (cb5r) catalyzes the reduction of the ferric iron (Fe3+) atom of the heme cofactor found within cytochrome b5 (cb5) by the reduction of the FAD cofactor of cb5r from reducing equivalents of the physiological electron donor, reduced nicotinamide adenine dinucleotide (NADH). Cb5r is characterized by the presence of two domains necessary for proper enzyme function: a flavin-binding domain and a pyridine nucleotide-binding domain. Within these domains are highly conserved "motifs" necessary for the correct binding and orientation of both the NADH coenzyme and the FAD cofactor. To address the importance of these conserved motifs, site-directed mutagenesis was utilized to generate a series of variants of residues located within the motifs to allow for the full characterizations. Second, naturally occurring recessive congenital methemoglobinemia (RCM) mutants found in proximity to these highly conserved motifs were analyzed utilizing site-directed mutagenesis. In addition, a canine variant of the cb5r soluble domain was cloned, generated and characterized and compared with the WT rat domain. The canine construct showed a high degree of sequence homology to that of the corresponding human and rat sequences. Characterization of the canine variant indicated that it possessed comparable functional characteristics to the rat variant. Investigation of the pyrophosphate-associating residues, Y112 and Q210, indicated that each played a role in the proper association and anchoring of NADH to the enzyme. The RCM type I mutants, T116S and E212K, caused a moderate decrease in efficiency of the enzyme. The presence of both mutations interact synergistically to generate a more substantially decreased function Analysis of the "180GtGitP185" NADH-binding motif and the preceding residue G179 revealed that these residues are vital in enabling proper NADH association. The residues of this motif were shown to be important in determining nucleotide specificity and properly positioning the NADH and flavin cofactor for efficient electron transfer. RCM variants A178T and A178V were shown to decrease catalytic efficiency or protein stability respectively, leading to disease phenotype. Analysis of the NADH-binding motif "273CGxxxM278" indicated that this motif facilitates electron transfer from substrate to cofactor and is important in release of NAD+ from the enzyme after electron transfer.

In order to provide an improved in vitro model with which to investigate human diseases, such as cancer that may be promoted by toxicant exposure, we have characterized a newly developed cell line derived from the renal proximal tubule epithelial cells (RPTEC) of a healthy human male donor. The RPTEC/TERT1 cell line has been immortalized using the human telomerase reverse transcriptase (hTERT) catalytic subunit and does not exhibit chromosomal abnormalities (Evercyte Laboratories). We have conducted single-compound and binary mixture experiments with the common environmental carcinogens, cadmium (Cd) and benzo[a]pyrene (B[a]P). Cells exhibited cytotoxicity to concentrations of B[a]P and Cd as low as 1 nM and 3 μM, respectively. We examined a panel of eight genes relevant to the toxic responses of these two agents. RPTEC/TERT1 cells exhibit compound-specific gene expression responses to concentrations as low as 1 nM B[a]P and 1 μM Cd. A significant increase in the expression of genes coding for B[a]P metabolizing enzymes (CYP1A1, CYP1B1) occurred in a dose- and time-dependent manner. Activity of these enzymes was verified using the EROD activity assay. Gene expression changes after co-exposure were consistent with changes in gene expression seen after single-compound exposures. We detected BPDE-DNA adducts after exposure to B[a]P which confirms that the RPTEC/TERT1 cell line responds to B[a]P consistently with what is known regarding these cells in a normal, healthy kidney. Under co-exposure, adducts detected were significantly decreased in some groups. A significant increase in the expression of NRF2 antioxidant pathway genes after co-exposure was observed. Additionally, total glutathione levels were significantly increased in cells exposed to Cd alone and co-exposure groups. These results suggest that Cd may antagonize the formation of BPDE-DNA adducts in RPTEC/TERT1 cells under these conditions. Future studies will test mutagenesis under conditions of co-exposure to Cd and B[a]P. Our studies are the first to provide information regarding toxicological responses in this novel cell line that model those of the target tissue. We conclude that these cells can provide a useful tool for future toxicological studies. These studies will help scientists better understand the initiating events that may promote carcinogenesis in normal, healthy human cells. / acase@tulane.edu

NADH:Cytochrome b5 Reductase (cb5r) catalyzes the two electron reduction of the iron center of the heme cofactor found within cytochrome b5 (cb5) utilizing reducing equivalents of the nicotinamide adenine dinucleotide (NADH) coenzyme. Cb5r is characterized by two domains necessary for proper enzyme function: a flavin-binding domain and a pyridine nucleotide-binding domain. Within these domains are highly conserved "motifs" necessary for the proper binding and orientation of both the NADH coenzyme and the FAD cofactor. To address the importance of these conserved motifs site-directed mutagenesis was utilized to generate a series of variants upon residues found within the motifs to allow for the full characterizations. Second, naturally occurring recessive congenital methemoglobinemia (RCM) mutants that are found within or in close proximity to these highly conserved motifs were analyzed utilizing site-directed mutagenesis. The flavin-binding motif "91RxYSTxxSN97" was characterized by the generation of variants T94H, T94G, T94P, P95I, V96S, and S97N. In addition to this, the naturally occurring double mutant P92H/E255- was fully characterized to establish a role of the P92 residue giving rise to RCM. The role of the "124GRxxST127" was determined by the introduction of a positive charge, charge reversal, and conserved amino acid mutations through site-directed mutagenesis of the G124, K125, and M126 residues. Based on the data presented here, each of the residues of the GRxxST motif are directly involved in maintaining the proper binding and orientation of the cb5r flavin prosthetic group. Analysis of the NADH-binding motif "273CGxxx-M278" was accomplished through the characterization of the type II RCM variant M272- and the type I RCM variant P275L. This demonstrates that the deletion of the M272 residue causes a frame shift leading to the inability of the NADH substrate to bind. The introduction of the P275L variant showed that substrate affinity was diminished, yet turnover was comparable to wild-type cytochrome b5 reductase, indicating that although P275 is required for proper substrate binding it is not essential for overall catalytic function. Finally, analysis of the naturally occurring double mutant G75S/V252M provided the first insight into a methemoglobinemia variant that possessed mutations in both the FAD-binding and NADH-binding domains.

Among the genetic mutations involved in carcinogenesis, TP53 mutation is a frequent event in many types of cancer. P53 is a transcription factor that regulates activities such as cell cycle arrest, apoptosis, DNA repair and angiogenesis. The majority of TP53 mutations are missense mutations that accumulate in cancer and are often retained in distant metastases. The effects of the mutant p53 proteins include loss of function, dominant-negative effects over wild-type (WT) p53 and possible acquisition of new properties (gain-of-function). However, some of these properties may differ from one mutant p53 protein to another. These differences could have implications for the in vivo behaviour of tumours carrying particular mutations and hence patient prognosis. The aim of this project was to investigate the phenotypic variation between cells transformed with different p53 mutants. This was achieved by constructing a range of TP53 mutants (R175H, G245S, R248W, R248Q, R273H, R282W) using PCR-based mega-primer site directed mutagenesis. These mutants were cloned into a mammalian bi-cistronic expression vector (designed for the co-expression of WT and mutant TP53 from a single plasmid) to allow transient expression in NCI-H358 cells (p53 null). Regard to the method for PCR site directed mutagenesis, the main technical difficulty with conventional methods was the insufficiency of the mutant TP53 product yield (75%). This thesis has modified these methods by carrying over the start template to a second round of PCR and increasing the MgCl2 concentration. This modified PCR-based site directed mutagenesis method has demonstrated an increased mutant TP53 product yield (100%). The tetracycline expression system is the most widely used for conditional inducible systems in mammalian cells, although high background expression has been a main problem. The ecdysone inducible system potentially allows for the study of the conditional expression of the exogenous reporter gene even though it may be cell lethal or alter the phenotype during the selection of transfectants. This system relies on two independent transfections of two plasmids namely pVgRXR and pIND. However, disruption of the regulatory element within the plasmid during stable integration can result in silence or high background expression of the exogenous reporter gene. A previous study reported a transient luciferase reporter assay to screen the cell line stably transfected with pVgRXR plasmid. However, there is no suitable method to screen the subsequent pIND transfection. This thesis has demonstrated a real time RT-PCR strategy to screen for the background expression problem associated with the ecdysone expression system. However, due to the project’s time limitations, a transient expression system rather than a stable expression system was used. The metastasis related cellular activity of WT/mutant TP53 transfected NCI-H358 cells was examined using a range of in vitro functional assays including a proliferation assay, a p21 promoter binding activity assay, a colony formation assay, and a migration assay. To extend the study, this thesis also employed real-time RT-PCR to examine the mRNA expression level of three metastatic related genes, VEGF, HER-2, and E-cadherin, in the WT/mutant TP53 transfected NCI-H358 cells. The results showed that different WT/mutant TP53 transfected cell linse could contribute to markedly different cellular activity. Among these mutants, R175H produced the highest cellular proliferation activity, the strongest dominant-negative activity over the WT on the p21 promoter binding activity and apoptosis activity, and the greatest effect on cellular migration. Furthermore, the real-time PCR results showed that the WT p53 inhibited transcription of key metastasis-related genes such as VEGF and HER-2. Considered with recent literature, this led me to postulate a feedback amplification cycle involving defective p53 and HER-2 mRNA expression. In conclusion, cancer cells with the R175H mutant could contribute to aggressive tumours. This conclusion, based on the in vitro data, is consistent with some clinical observations and animal model experiments. In the past few years it has become apparent that epigenetic changes also play a vitally important role in the cancer developmental process. Recent studies have reported the p53 protein can contribute in methylation which is one of the processes involved in epigenetic modification. This thesis employed a very new PCR-based AMP technique to examine the change of the global genome methylation pattern as a result of knocked-out p53 protein. The results showed defective p53 protein expression may associate with the global genome methylation pattern changes. However, it is important to note that antibiotic reagents, which were used for stable transfectant selection, could also contribute to the global genome methylation changes. In conclusion, this thesis has successfully developed two new methods. One allows the generation of a genetic mutant construct using PCR-based site directed mutagenesis while the other screens the tightly regulated ecdysone reporter system. In terms of effect of p53 in in vitro cell activity, this thesis has postulated that the R175H mutation is associated with much more aggressive metastatic cellular activity. Finally, this thesis also reported that loss of p53 expression could also result in changes in the global genome methylation pattern.

RNA metabolism includes all the processes required for RNA synthesis, maturation, and degradation in living cells. Ribonucleases (RNases) are involved in RNA maturation and degradation, two essential processes in gene expression and regulation in both prokaryotes and eukaryotes. Oligoribonuclease (Orn) has an important role in eliminating small oligonucleotides (nano-RNA), the last step in mRNA degradation. In E. coli, Orn is the only essential exoribonuclease. The enzyme has been shown to form a stable dimer, both in solution and in the crystalline form. Analysis of the three-dimensional structure of Orn allowed us to hypothesize that dimerization is essential for enzyme catalysis. In order to test the hypothesis, I analyzed a number of deletion and point mutants of Orn and determined that tryptophan 143 is essential for dimerization. A W143A mutant is unable to dimerize and has very little activity, similar to that of an active site mutant (D162A). The atomic structure of the W143A mutant, solved at a resolution of 1.9 Å, showed that although the overall three-dimensional fold is similar to that of the wild-type protein, minor differences exist that could account for the monomeric behavior in solution. A flexible Arg174 is repositioned into the cavity created by the missing Trp143. In this new orientation Arg174 protrudes into a hydrophobic pocket in the dimerization interface and is proposed to produce sufficient unfavorable interactions to keep the monomers apart in solution. All these data suggest that dimerization of Orn is essential for its activity. The human homolog of Orn, also known as small fragment nuclease (Sfn), has been shown to degrade short single-stranded RNA, the last step in mRNA decay. In order to determine the mechanism of action of Sfn and its role in the cell, we solved the crystal structure of a truncated form of Sfn at a resolution of 2.6 Å. This mutant form of Sfn lacks the C-terminal 21 amino acids (Sfn-∆C21) yet is as efficient as full length Sfn on model substrates. Interestingly, Sfn is not as active as E. coli Orn in in vitro assays. Analysis of the atomic structure revealed that the active site cleft in Sfn is narrower than the corresponding active site in E. coli. We propose a model for how this narrower cleft may explain the lower in vitro activity. Bacillus subtilis does not have an Orn homolog and until recently, the enzyme responsible for nano-RNA degradation in this organism was unknown. YtqI (also termed nrnA or nanoRNase), a protein unrelated to E. coli Orn, was recently shown to be responsible for the digestion of oligonucleotides in B. subtilis. In order to better understand the mechanism of action of YtqI, I solved its crystal structure at a resolution of 2.0 Å. The nuclease has a RecJ-like fold with two globular domains connected via a flexible linker that forms a central groove. On one side of the groove, the larger N-terminal domain harbors the putative active site, while on the opposite side, the C-terminal domain includes a putative RNA binding domain. The structure of YtqI provides insights into how this enzyme binds and digests oligoribonucleotides. The studies described here provide a better understanding of the mechanism of action for several exoribonucleases that act on nano-RNA oligonucleotides - Oligoribonuclease from E. coli, its close homolog in humans (Small fragment nuclease), as well as a functional homolog in Bacillus (YtqI). This work is relevant to understanding RNA metabolism, which is an essential process for survival of both eukaryotic and prokaryotic organisms.

Undergraduate honors paper--Mount Holyoke College, 2006. Dept. of Biological Sciences. / Includes bibliographical references (leaves 80-84).

<p><i>Lactococcus lactis,</i> prolidase (<i>Lla</i>prol) hydrolyzes Xaa-Pro dipeptides. Since Xaa-Pro is known as bitter peptides, <i>Lla</i>prol is potentially applicable to reduce bitterness of fermented foods. <i>Lla</i>prol shows allosteric behaviour and substrate inhibition, which are not reported in other prolidases. Computer models of <i>Lla</i>prol based on an X-ray structure of non-allosteric <i>Pyrococcus furiosus</i> prolidase showed that a loop structure (Loop^32-43) is located at the interface of the protomers of this homodimeric metallodipeptidase. This study investigated roles of four charged residues (Asp³⁶, His³⁸, Glu³⁹, and Arg⁴⁰) of Loop^32-43 in <i>Lla</i>prol using a combination of kinetic examinations of ten mutant enzymes and their molecular models. Deletion of the loop structure by Î36-40 mutant resulted in a loss of activity, indicating Loop^32-43 is crucial for the activity of <i>Lla</i>prol. D36S and H38S exhibited 96.2 % and 10.3 % activity of WT, whereas little activities (less than 1.0 % of WT activity) were observed for mutants E39S, D36S/E39S, R40S, R40E, R40K and H38S/R40S. These results implied that Glu³⁹ and/or Arg⁴⁰ play critical role(s) in maintaining the catalytic activity of <i>Lla</i>prol. These observations suggested that the loop structure is flexible and this attribute, relying on charge-charge interactions contributed by Arg⁴⁰, Glu³⁹ and Lys¹⁰⁸, is important in maintaining the activity of <i>Lla</i>prol. When the loop takes a conformation close to the active site (closed state), Asp³⁶ and His³⁸ at the tip of the loop can be involved in the catalytic reaction of <i>Lla</i>prol. The two active mutant prolidases (D36S and H38S) resulted in modifications of the unique characteristics; the allosteric behaviour was not observed for D36S, and H38S <i>Lla</i>prol showed no substrate inhibition. D36E/R293K, maintaining the negative charge of position 36 and positive charge of position 293, still possessed the allosteric behaviour, whereas the loss of the charges at these positions (D36S of this study and R293S of a previous study (Zhang et al., 2009 BBA-Proteins Proteom 1794, 968-975) eliminated the allosteric behaviour. These results indicated the charge-charge attraction between Asp³⁶ and Arg²⁹³ is important for the allostery of <i>Lla</i>prol. In the presence of either zinc or manganese divalent cations as the metal catalytic centre, D36S and H38S enzymes also showed different substrate preferences from WT <i>Lla</i>prol, implying the influence of Asp³⁶ and His³⁸ on the substrate binding. D36S and H38S also showed higher activities at pH 5.0 to 6.0, in which range WT <i>Lla</i>prol steeply decreased its activity, indicating Asp³⁶ and His³⁸ are involved in the active centre and influence the microenvironment of catalytic His²⁹⁶. The above observations are attributed to modifications of their local structure in the active centre since the temperature dependency and thermal denaturing temperature indicated little effects on the overall structure of the <i>Lla</i>prol mutants.</p> <p>From these results, we concluded that the unique behaviours of <i>Lla</i>prol are correlated to Loop^32-43 and Asp³⁶ and His³⁸ on it. When Loop^32-43 takes a closed conformation, Asp³⁶ interacts with Arg²⁹³ via charge-charge attraction to form an allosteric subsite. The saturation of the allosteric site with substrates further allowed the communications of His³⁸ with S₁ site residues to complete the active site. When the substrate concentration becomes higher than it is required to saturated productive S₁' site, His³⁸, Phe¹⁹⁰ and Arg²⁹³ would resemble the residue arrangement of S₁' site residues (His²⁹², Tyr³²⁹, and Arg³³⁷) and bind to the proline residue of substrates. This non-productive binding would prevent the conformational change of Loop^32-43, which further results in the substrate inhibition. For further confirmation of this mechanism, crystallographic studies will be conducted. In this thesis, we have indentified the conditions to produce crystals of <i>Lla</i>prol proteins.</p>