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MECHANICAL ABRASION AND ELECTROPORATION IN THE TRANSFORMATION OF INTACT PLEUROTUS OSTREATUS HYPHÆ and NUTRIENT-DEPENDANT RESISTANCE TO HYGROMYCIN BCordesman, Alexander 01 January 2008 (has links)
ABSTRACT 1: Abrasive treatment and subsequent electroporation of the basidiomycete Pleurotus ostreatus in a hypertonic buffer was investigated as a potential method of transforming of intact hyphæ. Mycelia, which are not capable of being transformed via electroporation due to interference by the cell wall, were abraded in an attempt to mechanically degrade the cell wall prior to electroporation. An electroporation field strength of 12,500 V/cm for 500 μs to 1.25 ms was found to be optimal based upon mortality effects. A 32 μm carborundum abrasive was initially evaluated but was quickly found to be inappropriately large so a corundum abrasive with an average size of 300 nm was focused on. Vortexing as well as low and medium power ultrasonic agitation with the corundum abrasive were investigated for their potential to cause mechanical degradation of the cell walls. Vortexing and low power sonication were found to be ineffective at causing adequate degradation while medium power sonication was found to be both ineffective and super lethal. While the possibility of mechanical abrasion facilitating transformation via electroporation remains, it is unlikely that conventional methods of agitation will be effective. ABSTRACT 2: The dose response of the basidiomycetes Pleurotus ostreatus, Agrocybe aegerita and Cordyceps millitaris to the antibiotic hygromycin B was tested on two common and one in-house solid growth media. The three species were grown on Potato dextrose agar, malt extract agar and an agar containing maltose, yeast-extract, peptone and glucose with concentrations of hygromycin B from zero to 100 μg/ml for 11 to 14 days. Micrometer measurements were used to determine the growth rate of each species on each media. Significant differences in hygromycin B tolerance for each species between the three media types were evident (p-value < .0001 by ANOVA for all). Neither the media type nor the growth rate on hygromycin B free plates were useful predictors of effective hygromycin B doses so optimization should be performed on every strain and media type used for selection.
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Crystallographic characterization of the ribosomal binding site and molecular mechanism of action of Hygromycin A.Kaminishi, Tatsuya, Schedlbauer, Andreas, Fabbretti, Attilio, Brandi, Letizia, Ochoa Lizarralde, Borja, He, Cheng-Guang, Milon, Pohl, Connell, Sean R, Gualerzi, Claudio O, Fucini, Paola 16 November 2015 (has links)
Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC. / Bizkaia:Talent and the European Union's Seventh Framework Program (Marie Curie Actions; COFUND; to S.C., A.S., T.K.); Marie Curie Actions Career Integration Grant (PCIG14-GA-2013-632072 to P.F.); Ministerio de Economía Y Competitividad (CTQ2014-55907-R to P.F., S.C.); FIRB Futuro in Ricerca from the Italian Ministero dell'Istruzione, dell'Universitá e della Ricerca (RBFR130VS5_001 to A.F.); Peruvian Programa Nacional de Innovación para la Competitividad y Productividad (382-PNICP-PIBA-2014 (to P.M. and A.F.)). Funding for open access charge: Institutional funding. / Revisión por pares
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Genetic Transformation of Switchgrass (Panicum Virgatum L.) with Endoglucanase Gene and Characterization of Plants with Endoglucanase TransgeneDere, Madhavi Suresh 24 August 2012 (has links)
As a warm season grass native to the North American continent, switchgrass is considered as one of the most promising biofuel crops in the USA. It is a C4 plant that makes it energy efficient. Switchgrass has a deep root system that allows it to grow on marginal land with low water and nutrient input. Switchgrass has been used as a forage crop and its use for biofuel will not affect food security. Biofuels are more environment-friendly than fossil fuels as they do not produce net greenhouse gases. However, the problem of high cost of production per unit for biofuel has to be overcome if we want to replace fossil fuels with biofuels. One of the major factors related to the high cost of biofuel are the expensive cellulase enzymes used in the pretreatment of feedstock. Endoglucanase is the key enzyme used for breaking down cellulose before fermentation. Currently, endoglucanase is produced from engineered E. coli or yeast strains, which is still expensive for enzyme production and purification of industrial scales. Expression of endoglucanase in plants has been previously reported. However, there are no reports of transgenic switchgrass producing cellulase enzyme. In this study, the catalytic domain of beta-endoglucanase gene was codon-optimized and synthesized based on the cDNA cloned from Hypocrea jecorina. Rice RuBisCO small subunit targeting signal peptide was fused to the N-terminus of the beta-endoglucanase gene, which was expected to target the fusion protein to chloroplast. This subcellular compartment targeting could minimize negative effects on cell function and plant development. The endoglucanase gene was cloned with maize ubiquitin promoter in a modified binary vector pCambia 1305-2 and transformed into switchgrass genotype HR8 by using Agrobacterium tumefaciens. In this study, I generated five independent transgenic switchgrass lines and they were confirmed by growing on the selection agent hygromycin, GUS assay, PCR amplification, southern blotting hybridization, for the presence of hygromycin and endoglucanase genes. However, based on RT-PCR analysis, only two transgenic lines were confirmed to produce mRNAs of the endoglucanase gene. These two transgenic lines were further characterized for their agronomic traits and the chlorophyll contents. Our results suggested that expression of endoglucanase in switchgrass could reduce chlorophyll content and affect plant development. Nevertheless, in this study, we demonstrated that a fungal endoglucanase gene could be expressed in switchgrass transgenic plants, though the gene expression level and the subcellular localization need to be carefully regulated in order to minimize the toxic effect of endoglucanase on plant cells. / Master of Science
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Modelagem molecular na caracteriza??o eletr?nica de oligopept?deos e na descri??o qu?ntica da intera??o f?rmaco-receptorOliveira, Jonas Ivan Nobre 02 March 2012 (has links)
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Previous issue date: 2012-03-02 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / In this dissertation, the theoretical principles governing the molecular modeling were applied for
electronic characterization of oligopeptide α3 and its variants (5Q, 7Q)-α3, as well as in the quantum
description of the interaction of the aminoglycoside hygromycin B and the 30S subunit of bacterial
ribosome. In the first study, the linear and neutral dipeptides which make up the mentioned
oligopeptides were modeled and then optimized for a structure of lower potential energy and
appropriate dihedral angles. In this case, three subsequent geometric optimization processes, based
on classical Newtonian theory, the semi-empirical and density functional theory (DFT), explore the
energy landscape of each dipeptide during the search of ideal minimum energy structures. Finally,
great conformers were described about its electrostatic potential, ionization energy (amino acids),
and frontier molecular orbitals and hopping term. From the hopping terms described in this study, it
was possible in subsequent studies to characterize the charge transport propertie of these peptides
models. It envisioned a new biosensor technology capable of diagnosing amyloid diseases, related to
an accumulation of misshapen proteins, based on the conductivity displayed by proteins of the
patient. In a second step of this dissertation, a study carried out by quantum molecular modeling of
the interaction energy of an antibiotic ribosomal aminoglicos?dico on your receiver. It is known that
the hygromycin B (hygB) is an aminoglycoside antibiotic that affects ribosomal translocation by direct
interaction with the small subunit of the bacterial ribosome (30S), specifically with nucleotides in
helix 44 of the 16S ribosomal RNA (16S rRNA). Due to strong electrostatic character of this
connection, it was proposed an energetic investigation of the binding mechanism of this complex
using different values of dielectric constants (ε = 0, 4, 10, 20 and 40), which have been widely used to
study the electrostatic properties of biomolecules. For this, increasing radii centered on the hygB
centroid were measured from the 30S-hygB crystal structure (1HNZ.pdb), and only the individual
interaction energy of each enclosed nucleotide was determined for quantum calculations using
molecular fractionation with conjugate caps (MFCC) strategy. It was noticed that the dielectric
constants underestimated the energies of individual interactions, allowing the convergence state is
achieved quickly. But only for ε = 40, the total binding energy of drug-receptor interaction is
stabilized at r = 18A, which provided an appropriate binding pocket because it encompassed the
main residues that interact more strongly with the hygB - C1403, C1404, G1405, A1493, G1494,
U1495, U1498 and C1496. Thus, the dielectric constant ≈ 40 is ideal for the treatment of systems
with many electrical charges. By comparing the individual binding energies of 16S rRNA nucleotides
with the experimental tests that determine the minimum inhibitory concentration (MIC) of hygB, it is
believed that those residues with high binding values generated bacterial resistance to the drug
when mutated. With the same reasoning, since those with low interaction energy do not influence
effectively the affinity of the hygB in its binding site, there is no loss of effectiveness if they were
replaced. / Nessa disserta??o, os princ?pios te?ricos que regem a Modelagem Molecular foram aplicados na
caracteriza??o eletr?nica do oligopept?deo α3 e seus variantes (5Q,7Q)-α3, como tamb?m na
descri??o qu?ntica da intera??o do aminoglicos?deo higromicina B e a subunidade 30S do ribossomo
bacteriano. No primeiro estudo, os dipept?deos lineares e neutros constituintes das biomol?culas
mencionados foram modelados e posteriormente otimizados at? uma estrutura de menor energia
potencial e ?ngulos diedros adequados. No caso, tr?s processos de otimiza??o geom?trica, baseados
subsequentemente na teoria cl?ssica newtoniana, na semi-emp?rica e na teoria do funcional da
densidade (DFT), varreram a paisagem de energia de cada dipept?deos na busca de uma estrutura de
energia m?nima ideal. Por fim, os conf?rmeros ?timos foram descritos quanto ao potencial
eletrost?tico, energia de ioniza??o (amino?cidos), orbitais de fronteira HOMO/HOMO-1 e termo de
hopping. A partir dos termos de hopping descritos nesse trabalho, foi poss?vel, em estudos
subsequentes, caracterizar as propriedades de transporte de cargas destes modelos pept?dicos.
Vislumbra-se uma nova tecnologia de biosensores capaz de diagnosticar doen?as amiloides,
relacionadas ao ac?mulo de pept?deos disformes, a partir do perfil de condutividade el?trica
apresentado pelas prote?nas do paciente. Em um segundo momento dessa disserta??o, realiza-se um
estudo qu?ntico por modelagem molecular da energia de intera??o de um antibi?tico
aminoglicos?dico em seu receptor riboss?mico. Sabe-se que a higromicina B (higB) ? um antibi?tico
aminoglicos?deo que afeta a transloca??o ribossomal pela intera??o direta com a subunidade menor
do ribossomo bacteriano (30S), especificamente com nucleot?deos da h?lice 44 do RNA riboss?mico
16S (rRNA 16S). Devido ao forte car?ter eletrost?tico desta conex?o, foi proposta a investiga??o
energ?tica do mecanismo de liga??o da higB no 30S usando diferentes valores de constantes
diel?tricas (ε=0, 4, 10, 20 e 40), as quais s?o amplamente utilizadas no estudo das propriedades
eletrost?ticas de biomol?culas. Para isso, foram medidos raios crescentes centralizados no centr?ide
da higB tendo por base a estrutura cristalina higB-30S (1HNZ.pdb), e apenas a energia de intera??o
individual de cada nucleot?deo englobado foi calculada quanticamente utilizando a estrat?gia de
fracionamento molecular com capuzes conjugados (MFCC). Percebeu-se que as constantes
diel?tricas subestimam as energias de intera??o individuais, permitindo que o estado de
converg?ncia energ?tica seja alcan?ado rapidamente. Por?m apenas para ε=40, a energia de
intera??o total droga-receptor se estabilizou em r=18?, o que se constituiu como um adequado s?tio
de liga??o, pois englobou os res?duos do 16S que interagem mais fortemente com a higB - C1403,
C1404, G1405, A1493, G1494, U1495, C1496 e U1498. Assim, a constante diel?trica ≈40 ? ideal para
o tratamento de sistemas com muitas cargas. Confrontando as energias de liga??o individuais dos
nucleot?deos 16SrRNA com ensaios experimentais para determina??o da concentra??o inibit?ria
m?nima (MIC) da higB, acredita-se que esses res?duos com elevados valores de intera??o gerariam
resist?ncia bacteriana ? droga quando mutados. Com o mesmo racioc?nio, visto que aqueles com
baixa energia n?o influenciariam de forma eficaz a afinidade da higB em seu s?tio de liga??o, n?o
ocorreria perda de efic?cia caso fossem substitu?dos.
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