Reducing the production cost of industrial biofuels will greatly facilitate their proliferation and co-integration with fossil fuels. The cost of feedstock is the largest cost in most fermentation bioprocesses and therefore represents an important target for cost reduction. Meanwhile, the biorefinery concept advocates revenue growth through complete utilization of by-products generated during biofuel production. Taken together, the production of biofuels from low-cost crude glycerol, available in oversupply as a by-product of bioethanol production, in the form of thin stillage, and biodiesel production, embodies a remarkable opportunity to advance affordable biofuel development. However, few bacterial species possess the natural capacity to convert glycerol as a sole source of carbon and energy into value-added bioproducts. Of particular interest is the anaerobe Clostridium pasteurianum, the only microorganism known to convert glycerol alone directly into butanol, which currently holds immense promise as a high-energy biofuel and bulk chemical. Unfortunately, genetic and metabolic engineering of C. pasteurianum has been fundamentally impeded due to a complete lack of genetic tools and techniques available for the manipulation of this promising bacterium. This thesis encompasses the development of fundamental genetic tools and techniques that will permit extensive genetic and metabolic engineering of C. pasteurianum. We initiated our genetic work with the development of an electrotransformation protocol permitting high-level DNA transfer to C. pasteurianum together with accompanying selection markers and vector components. The CpaAI restriction-modification system was found to be a major barrier to DNA delivery into C. pasteurianum which we overcame by in vivo methylation of the recognition site (5’-CGCG-3’) using the M.FnuDII methyltransferase. Systematic investigation of various parameters involved in the cell growth, washing and pulse delivery, and outgrowth phases of the electrotransformation procedure significantly elevated the electrotransformation efficiency up to 7.5 × 104 transformants µg-1 DNA, an increase of approximately three orders of magnitude. Key factors affecting the electrotransformation efficiency include cell-wall-weakening using glycine, ethanol-mediated membrane solubilization, field strength of the electric pulse, and sucrose osmoprotection. Following development of a gene transfer methodology, we next aimed to sequence the entire genome of C. pasteurianum. Using a hybrid approach involving 454 pyrosequencing, Illumina dye sequencing, and single molecule real-time sequencing platforms, we obtained a near-complete genome sequence comprised of 12 contigs, 4,420,100 bp, and 4,056 candidate protein-coding genes with a GC content of 30.0%. No extrachromosomal elements were detected. We provide an overview of the genes and pathways involved in the organism’s central fermentative metabolism. We used our developed electrotransformation procedure to investigate the use of established clostridial group II intron biology for constructing chromosomal gene knockout mutants of C. pasteurianum. Through methylome analysis of C. pasteurianum genome sequencing data and transformation assays of various vector deletion constructs, we identified a new Type I restriction-modification system that inhibits transfer of vectors harboring group II intron gene knockout machinery. We designated the new restriction system CpaAII and proposed a recognition sequence of 5’-AAGNNNNNCTCC-3’. Overcoming restriction by CpaAII, in addition to low intron retrohoming efficiency, allowed the isolation of a gene knockout mutant of C. pasteurianum with a disrupted CpaAI Type II restriction system. The resulting mutant strain should be efficienty transformed with plasmid DNA lacking M.FnuDII methylation. Lastly, we investigated the use of plasmid-based gene overexpression and chromosomal gene downregulation to alter gene expression in C. pasteurianum. Using a β-galactosidase reporter gene, we characterized promoters corresponding to the ferredoxin and thiolase genes of C. pasteurianum and show that both promoters permitted high-level, constitutive gene expression. The thiolase promoter was then utilized to drive transcription of an antisense RNA molecule possessing complementarity to mRNA of our β-galactosidase reporter gene. Our antisense RNA system demonstrated 52-58% downregulation of plasmid encoded β-galactosidase activity throughout the duration of growth. In an attempt to perturb the central fermentative metabolism of C. pasteurianum and enhance butanol titers, we prepared several antisense RNA constructs for downregulation of 1,3-propanediol, butyrate, and hydrogen production pathways. The resulting downregulation strains are expected to exhibit drastically altered central fermentative metabolism and product distribution. Taken together, we have demonstrated that C. pasteurianum is amendable to genetic manipulation through the development of methods for plasmid DNA transfer and gene overexpression, knockdown, and knockout. Further, our genome sequence should provide valuable nucleotide sequence information for the application of our genetic tools. Thus, the genome sequence, electrotransformation method, and associated genetic tools and techniques reported here should promote extensive genetic manipulation and metabolic engineering of this biotechnologically important bacterium.

Numerous organisms produce ornately detailed inorganic structures (often known as shells) with features on length scales from the nanoscale to the microscale. One organism, commonly referred to as a diatom, originates from algae and is found throughout the oceans on Earth. These diatoms possess skeletal structures, frustules, made from silicon dioxide. This chemical makeup limits the number of possible applications for which these structures can be used. Using a series of gas displacement reactions, these frustules can be converted to other useful materials, such as magnesium oxide and titanium dioxide, while maintaining the features of the frustule template. In the current research, silicon dioxide frustules were converted to titanium dioxide replicas using method previously devised by our group. The titanium dioxide replicas were subjected to a hydrothermal reaction by exposing the replicas to an aqueous basic solution containing barium hydroxide to form barium titanate and barium strontium titanate replicas. The effects of reaction temperature, time, and solution composition on extent of conversion were examined. The conventional method of converting titanium dioxide to barium titanate, using a convection heating oven, was compared with a microwave assisted heating method to study the advantages of using microwave heating over convection heating.

Our objective was to address current cell source limitations in engineering pancreatic â-cells for the treatment of type 1 diabetes by investigating retroviral genetic modification of murine embryonic stem cells (mESC) with a murine stem cell virus (MSCV) encoding proendocrine transcription factor Neurogenin 3 (Ngn3). We found that expression of Ngn3 and the enhanced green fluorescent protein (eGFP) reporter gene were both significantly silenced in genetically modified mESCs. To overcome this obstacle and enhance the efficiency of retroviral gene transfer to mESCs in general, we employed a virus-polymer complexation method to deliver more transgenes to mESCs. Despite increased transgene delivery and integration in mESCs, transgene expression did not increase. Results suggest mESCs may be restricted in several steps of retrovirus transduction. We then investigated which steps of the virus lifecycle restrict efficient transduction of mESCs by using a recombinant MMuLV-derived retrovirus and a recombinant HIV-1-derived lentivirus to compare three major steps in the transduction of mESCs and NIH 3T3 cells - virus binding, virus integration, and transgene expression. We found that retroviruses and lentiviruses similarly bind 3 or 4-fold less efficiently to R1 mES cells than to NIH 3T3 fibroblasts. We also detected 3-fold fewer integrated retrovirus transgenes and 11-fold lower expression levels in NIH 3T3 cells, suggesting the primary limitation to retrovirus transduction may be low levels of transgene expression. In contrast we detected 10-fold fewer integrated lentivirus transgenes and 8-fold lower expression levels, suggesting lentivirus transduction may be limited by inefficient intracellular post-binding steps of transduction. We then investigated whether depletion of linker histone 1 in mESCs would alleviate silencing of retrovirus transgenes and improve gene transfer by transducing histone H1c, H1d, H1e triple null mESCs with different recombinant vectors. We found this did not improve viral gene transfer. This research is significant for improving protocols for gene transfer to ES cells and facilitating the use of modified ES cells in regenerative medicine.

DNA is the code for all life. Via intermediary RNA the information encoded by the genome is relayed to proteins executing the various functions in a cell. Together, this repertoire of inherently linked biological macromolecules determines all characteristics and features of a cell. Technological advancements during the last decades have enabled the pursuit of novel types of studies and the investigation of the cell and its constituents at a progressively higher level of detail. This has shed light on numerous cellular processes and on the underpinnings of several diseases. For the majority of studies focusing on nucleic acids, an amplification step has to be implemented before an analysis, scoring or interrogation method translates the amplified material into relevant biological information. This information can, for instance, be the genotype of particular SNPs or STRs, or the abundance level of a set of interesting transcripts. As such, amplification plays a significant role in nucleic acid assays. Over the years, a number of techniques – most notably PCR – has been devised to meet this amplification need, specifically or randomly multiplying desired regions. However, many of the approaches do not scale up easily rendering comprehensive studies cumbersome, time-consuming and necessitating large quantities of material.Trinucleotide threading (TnT) – forming the red thread throughout this thesis – is a multiplex amplification method, enabling simultaneous targeted amplification of several nucleic acid regions in a specific manner. TnT begins with a controlled linear DNA thread formation, each type of thread corresponding to a segment of interest, by a gap-fill reaction using a restricted trinucleotide set. The whole collection of created threads is subsequently subjected to an exponential PCR amplification employing a single primer pair. The generated material can thereafter be analyzed with a multitude of readout and detection platforms depending on the issue or characteristic under consideration.TnT offers a high level of specificity by harnessing the inherent specificities of a polymerase and a ligase acting on a nucleotide set encompassing three out of the four nucleotide types. Accordingly, several erroneous events have to occur in order to produce artifacts. This necessitates override of a number of control points.The studies constituting this thesis demonstrate integration of the TnT amplification strategy in assays for analysis of various aspects of DNA and RNA. TnT was adapted for expression profiling of intermediately-sized gene sets using both conventional DNA microarrays and massively parallel second generation 454 sequencing for readout. TnT, in conjunction with 454 sequencing, was also employed for allelotyping, defined as determination of allele frequencies in a cohort. In this study, 147 SNPs were simultaneously assayed in a pool comprising genomic DNA of 462 individuals. Finally, TnT was recruited for parallel amplification of STR loci with detection relying on capillary gel electrophoresis. In all investigations, the material generated with TnT was of sufficient quality and quantity to produce reliable and accurate biological information.Taken together, TnT represents a viable multiplex amplification technique permitting parallel amplification of genomic segments, for instance harboring polymorphisms, or of expressed genes. In addition to these, this versatile amplification module can be implemented in assays targeting a range of other features of genomes and transcriptomes. / QC 20100819

Kiwifruit are of huge economic importance for New Zealand representing 29 percent of total horticultural exports. Fruit size is the biggest determinant of what consumers are willing to pay, and there is also a positive relationship between consumer preference for flavour and percentage dry matter. The two main cultivars exported from New Zealand are Actinidia chinensis ‘Hort 16A’ (gold kiwifruit) and A. deliciosa ‘Hayward’ (green kiwifruit). Under current commercial practice the only product allowed for use on kiwifruit to increase fruit size in New Zealand is Benefit®. Benefit® has been shown to induce different results when applied to A. chinensis and A. deliciosa, whereas synthetic plant growth regulators such as the cytokinin-like substance N-(2- chloro-4-pyridyl)-N’-phenylurea (CPPU) have been found to promote similar increases in fresh weight of fruit in both cultivars. Final fruit size is determined by both cell division and cell enlargement. It was been shown that fresh weight can be increased in both of the major Actinidia cultivars even though their physiology differs. Hormonal control of fruit size in relation to cell division and cell enlargement phases of fruit growth was studied in both A. chinensis and A. deliciosa. CPPU was applied to both cultivars in a growth response experiment where fruit were collected throughout the growing season. The objective of this experiment was to create growth curves, to compare and contrast the effect on A. chinensis and A. deliciosa, and to provide material for hormone analysis. Application of CPPU was found to significantly increase the fresh weight of both A. chinensis and A. deliciosa fruit (46.98 and 31.34 g increases respectively), and alter the ratio of inner and outer pericarps of A. chinensis fruit. CPPU and Benefit® were applied individually and together to both cultivars. It was found that only A. chinesis fruit were affected by the application of Benefit®; fresh weight was increased by 26.38 g, and percentage dry matter was significantly reduced. There was a statistically significant (p < 0.05) interaction between CPPU and Benefit® when applied to A. chinensis. 3,5,6-trichloro-2-pyridyloxyacetic acid (3,5,6-TPA) was applied to A. deliciosa on two application dates at three concentrations and was found to decrease fresh weight of fruit, but significantly increase percentage dry matter regardless of application date or concentration. Lastly CPPU and 1-naphthalene acetic acid (NAA) were applied to A. deliciosa at two application dates and in all combinations. Application date affected the response to both a low concentration of CPPU and NAA. A synergistic interaction was observed when CPPU was applied early plus NAA late (CPPU early (4.53 g increase) plus NAA late (13.29 g) < CPPU early plus NAA late (33.85 g). Finally endogenous hormone content was studied. Methods were developed and tested for the simultaneous analysis of both indole-3-acetic acid (IAA) and cytokinins. Freeze dried fruit were purified using Waters Sep-pak® cartridges and Oasis® columns then IAA was quantified by high pressure liquid chromatography. Preliminary results indicate a correlation between application of CPPU and endogenous IAA, high concentrations of IAA correlated well with periods of rapid fruit growth particularly for CPPU treated fruit.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / Worldwide, the rust diseases cause significant annual wheat yield losses (Wallwork 1992; Chrispeels & Sadava 1994). The utilization of host plant resistance to reduce such losses is of great importance particularly because biological control avoids the negative environmental impact of agricultural chemicals (Dedryver et al. 1996). The wild relatives of wheat are a ready source of genes for resistance to disease and insect pests. A large degree of gene synteny still exists among wheat and its wild relatives (Newbury & Paterson 2003). It is therefore possible to transfer a chromosome segment containing useful genes to a homologous region in the recipient genome without serious disruption of genetic information. Special cytogenetic techniques are employed to transfer genes from the wild relatives to the wheat genomes (Knott 1989). Unfortunately the transfer of useful genes may be accompanied by the simultaneous transfer of undesirable genes or redundant species chromatin which has to be mapped and removed (Feuillet et al. 2007). DNA markers are extremely useful for the characterisation and shortening of introgressed regions containing genes of interest (Ranade et al. 2001), and may also be used for marker aided selection of the resistance when the genes are employed commercially. Eight wheat lines containing translocations/introgressions of wild species-derived resistance genes were developed by the Department of Genetics (SU). These lines are presently being characterized and mapped and attempts are also being made to shorten the respective translocations. This study aimed to find DNA markers for the various translocations and to convert these into more reliable SCAR markers that can be used in continued attempts to characterize and improve the respective resistance sources. A total of 260 RAPD and 21 RGAP primers were used to screen the eight translocations and, with the exception of Lr19, it was possible to identify polymorpic bands associated with each translocation. However, it was not possible to convert all of these into more reliable SCAR markers. The primary reason for this was the low repeatability of most of the bands. Certain marker fragments turned out to be repeatable but could not be converted successfully. Some of the latter can, however, be used directly (in RAPD or RGAP reactions) as markers. The Lr19 translocation used in the study (Lr19-149-299) is a significantly reduced version of the original translocation and failure to identify polymorphisms associated with it can probably be ascribed to its small size. The following numbers of markers (direct and converted into SCARs) were Worldwide, the rust diseases cause significant annual wheat yield losses (Wallwork 1992; Chrispeels & Sadava 1994). The utilization of host plant resistance to reduce such losses is of great importance particularly because biological control avoids the negative environmental impact of agricultural chemicals (Dedryver et al. 1996). The wild relatives of wheat are a ready source of genes for resistance to disease and insect pests. A large degree of gene synteny still exists among wheat and its wild relatives (Newbury & Paterson 2003). It is therefore possible to transfer a chromosome segment containing useful genes to a homologous region in the recipient genome without serious disruption of genetic information. Special cytogenetic techniques are employed to transfer genes from the wild relatives to the wheat genomes (Knott 1989). Unfortunately the transfer of useful genes may be accompanied by the simultaneous transfer of undesirable genes or redundant species chromatin which has to be mapped and removed (Feuillet et al. 2007). DNA markers are extremely useful for the characterisation and shortening of introgressed regions containing genes of interest (Ranade et al. 2001), and may also be used for marker aided selection of the resistance when the genes are employed commercially. Eight wheat lines containing translocations/introgressions of wild species-derived resistance genes were developed by the Department of Genetics (SU). These lines are presently being characterized and mapped and attempts are also being made to shorten the respective translocations. This study aimed to find DNA markers for the various translocations and to convert these into more reliable SCAR markers that can be used in continued attempts to characterize and improve the respective resistance sources. A total of 260 RAPD and 21 RGAP primers were used to screen the eight translocations and, with the exception of Lr19, it was possible to identify polymorpic bands associated with each translocation. However, it was not possible to convert all of these into more reliable SCAR markers. The primary reason for this was the low repeatability of most of the bands. Certain marker fragments turned out to be repeatable but could not be converted successfully. Some of the latter can, however, be used directly (in RAPD or RGAP reactions) as markers. The Lr19 translocation used in the study (Lr19-149-299) is a significantly reduced version of the original translocation and failure to identify polymorphisms associated with it can probably be ascribed to its small size. The following numbers of markers (direct and converted into SCARs) were v identified: S8-introgression (Triticum dicoccoides) = one RAPD and two SCARs; S13-translocation (Aegilops speltoides) = four RAPDs, three RGAPs and five SCARs; S15-translocation (Ae. peregrina) = one RAPD and two SCARs; S20-translocation (Ae. neglecta) = two RAPDs, two RGAPs and one SCAR. The markers are already being employed in current projects aiming to map and shorten these translocations. Some of the markers can be combined in multiplex reactions for more effective mass screening. No repeatable markers could be identified for the four remaining translocations (S12 from Ae. sharonensis; S14 from Ae. kotschyi; Smac from Ae. biuncialis and Lr19-149-299 from Thinopyrum ponticum).

Made available in DSpace on 2015-03-26T13:32:22Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1444636 bytes, checksum: 6eeadcc5d1f7aefe015645aed7d241f2 (MD5) Previous issue date: 2014-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Until then, how to increase the gain by selection has been pointed out by different strategies recommended in breeding methods, or by using the basic principles of experimentation, or even use biometric models that seek to parameterize both genotypic and environmental influences. Thus, as regards the methods of genetics and statistics used for selection of superior genotypes, highlight the methods derived from the theory of direct selection, indirect selection and index selection, combined selection method and REML-BLUP method among others. Despite several methodologies available for the selection of superior genotypes is still common and necessary practice selection of individuals in relation to features with low reliability of predicting genotype value from a phenotypic value given by the corrected or adjusted by the aggregate average phenotypic information from relatives of correlated traits or practices to reduce the environmental effect. In the end, we consider this phenotypic average adjusted as the most appropriate measure to indicate genetic superiority and predict genetic gain. However, such models or procedures do not include a multitude of other statistical information of great relevance, different phenotypic average which is usually adopted, but which provide important information about the reported genotype and who have been sidelined studies involving genetic improvement and selection criteria. In this context, artificial neural networks are a new paradigm that has been employed, albeit tenuously, in animal and plant breeding programs. This approach, unlike the stochastic modeling used so far, is based on principles of learning and computational intelligence on a wide range of performance information of genotype involving averages, maximum, minimum, variance and all sorts of information can be directly or indirectly measured. Thus, unlike the statistical methods that summarize the information or perform the structural simplification of data, neural networks, like the human brain, capture all available information to generate a criterion for decision making. This work was carried out with the intention of using neural networks to improve the accuracy of the predicted values and genetic gains, through a discussion of its theoretical foundations and use simulated data with the same characteristics in terms of average heritability and coefficient of variation of actual data by providing an alternative method for identification of superior genotypes. / Até então, as formas de aumentar o ganho por seleção tem sido apontadas pelas diferentes estratégias preconizadas nos métodos de melhoramento, ou pela utilização dos princípios básicos da experimentação, ou ainda recorrer a modelos biométricos que buscam parametrizar as influências tanto genotípicas quanto ambientais. Assim, no que se refere aos métodos de genética e estatística utilizados para seleção de genótipos superiores, destacam-se os métodos derivados da teoria de seleção direta, seleção indireta e por índice de seleção, o método de seleção combinada e o método REML-BLUP, dentre outros. Apesar das diversas metodologias disponíveis para a seleção de genótipos superiores, ainda é comum e necessário praticar seleção de indivíduos em relação a características com baixa confiabilidade de predição do valor genotípico a partir de um valor fenotípico dado pela média fenotípica corrigida ou ajustada em função da agregação de informações de parentes, de caracteres correlacionados ou de práticas de redução do efeito ambiental. No final, considera-se esta média fenotípica ajustada como a medida mais apropriada para indicar a superioridade genética e predizer o ganho genético. No entanto, tais modelos ou procedimentos não contemplam uma infinidade de outras informações estatísticas de grande relevância, diferentes da média fenotípica que é usualmente adotada, mas que agregam informações importantes acerca do genótipo avaliado e que têm sido deixadas à margem dos estudos envolvendo melhoramento genético e critérios de seleção. Neste contexto, as redes neurais artificiais constituem novo paradigma que tem sido empregado, ainda que de forma tênue, nos programas de melhoramento genético animal e vegetal. Essa abordagem, diferentemente das modelagens estocásticas utilizadas até então, é baseada nos princípios de aprendizado e de inteligência computacional de um conjunto amplo de informação do desempenho do genótipo envolvendo médias, máximos, mínimos, variância e toda ordem de informação possível de ser direta ou indiretamente mensurada. Assim, ao contrário dos métodos estatísticos que resumem as informações ou realizam a simplificação estrutural dos dados, as redes neurais, à semelhança do cérebro humano, captam toda informação disponível para gerar um critério de tomada de decisão. Assim, este trabalho foi realizado com o intuito de utilizar as redes neurais para melhorar a acurácia na predição de valores e ganhos genéticos, através de uma discussão de seus fundamentos teóricos e utilização de dados simulados, com mesma caracterização em termos de média, herdabilidade e coeficiente de variação dos dados reais, fornecendo um método alternativo para identificação de genótipos superiores.

La protéine DnaA, ou un homologue, est présente dans la plupart des organismes vivants parce qu'elle joue un rôle clé pour la réplication de l'ADN. Dans Escherichia coli, l'expression de DnaA, l'initiateur central de la réplication de l'ADN, est donc étroitement régulée. Des études antérieures ont montré qu'une forte surexpression de cette protéine conduit à une diminution de la viabilité cellulaire, alors que son absence induit l'arrêt de la division cellulaire. Même si ces conditions extrêmes sont bien étudiées, la transition entre l'arrêt de la division et la croissance normale n'a pas été analysée quantitativement.Nous avons modifié génétiquement Escherichia coli pour mettre l'expression de l'ARN polymérase et de DnaA sous le contrôle deux systèmes inductibles distincts. Pour contrôler l’expression de DnaA, nous avons utilisé un système d’induction se trouvant déjà dans la cellule, le système uhp. Le promoteur du gène uhpT est induit par le glucose-6-phosphate extracellulaire. Nous avons tout d’abord étudié les caractéristiques d’induction de ce système et ensuite caractérisé les phénomènes biologiques déclenchées par les variations de la concentration de DnaA. Les méthodes utilisées combinent des mesures sur une population des bactéries, avec celles en cellule unique, en utilisant la microscopie in vivo en temps réel et des systèmes microfluidiques. Les expériences de microscopie révèlent des phénomènes stochastiques en raison du faible nombre de molécules des composants du système d'induction uhp. En corrélant les observations de population et de cellules uniques, nous donnons une interprétation quantitative du comportement observé. Comme une application potentielle de notre système de contrôle, nous envisageons la possibilité d’arrêter la division cellulaire afin de transformer la cellule en un «sac d'enzymes» pour la production biotechnologique de métabolites. / The DnaA protein, or a homologue, is present in most living organisms because it plays a key role for DNA replication. In Escherichia coli, the expression of DnaA, the central initiator of DNA replication, is therefore tightly regulated. Previous studies have shown that a large overexpression of this protein leads to a decrease in cell viability, while its absence induces the arrest of cell division. Even though these extreme conditions are well studied, the transition from division arrest to normal growth has not been quantitatively analyzed.We genetically engineered Escherichia coli to put the expression of RNA polymerase and the expression of DnaA under the control of two distinct, inducible systems. For the control of DnaA expression, we used a regulatory system already present in the cell, the uhp system. The promoter of the uhpT gene is induced via extracellular glucose-6-phosphate. We characterized the induction characteristics of this system and studied the biological phenomena triggered by varying concentrations of DnaA, using population measurements and single cell, time lapse microscopy of microcolonies or cells grown in a microfluidics device. The microscopy experiments reveal stochastic phenomena due to the low number of molecules of components of the induction system and of DnaA. Confronting population and single cell observations we are able to give a quantitative interpretation of the observed behavior. As a potential application of our control system, we explored the possibility of freezing cell division in order to turn the cell into a “bag of enzymes” for the biotechnological production of metabolites.

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Made available in DSpace on 2016-12-08T16:24:24Z (GMT). No. of bitstreams: 1 PGCV06MA014.pdf: 2536288 bytes, checksum: 2ced4e5eb18884533f8a66a86da4f665 (MD5) Previous issue date: 2006-07-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Aujeszky s disease (AD) is an infect-contagious illness that causes serious economical damages to the producer and the swine industry. Aiming to develop mechanisms and to improve technologies that are faster, more sensitive and more specific for diagnosis and for use in free areas or in AD s eradication programs, the sequence codifier of the glycoprotein E (gE) of Aujeszky s disease virus (ADV) was amplified, cloned and expressed. Through genetic engineering the sequence of the gE gene was propagated in an host organism. The gE was amplified by the technique of polimerase chain reaction (PCR), cloned in the vector pGem®-T Easy and transformed in competent cells of Escherichia coli, DH-5a . The clone obtained was sub-cloned in the expression plasmid pFastBac 1, which contains the promoter gene of the polyhedrin. The obtained subclone was analyzed inside for certification of its correct plasmid orientation with the restriction endonucleases BamH I and EcoR I. The sub-clone with the correct orientation had its DNA extracted and used for transposition inside the bacmid (recombinant baculovirus and helper plasmid with competent DH10Bac cell). Colonies with inserted gE were selected by the phenotype of the colony, which expresses white color when cloned. White recombinant colonies had their DNA extracted and used for cotransfection in insect cells Trichoplusia ni (BTI-Tn5B1-4). The recombinant-gE baculovirus was inoculated in cultured cells and expressed the recombinant gE by PCR and Western blotting . The recombinant-gE baculovirus containing only the gE gene of the VDA will be used for antigen and monoclonal antibodies production, which will aid in the development of a more sensitive, specific and safer for the use in VDA free regions / A doença de Aujeszky (DA) é uma enfermidade infecto-contagiosa que causa graves prejuízos econômicos ao produtor e à agroindústria suinícola. Com o objetivo de desenvolver insumos e aprimorar tecnologias que sejam mais rápidas, sensíveis e específicas de diagnóstico para uso em regiões livres ou em erradicação da DA, a seqüência codificadora da glicoproteína E (gE) do vírus da doença de Aujeszky (VDA) foi amplificada, clonada e expressada. Através da engenharia genética a seqüência do gene da gE foi propagada em um organismo hospedeiro. A gE foi amplificada pela técnica da reação em cadeia da polimerase (PCR) , clonada no vetor pGem®-T Easy e transformada em células competentes de Escherichia coli, DH-5a . O clone obtido foi subclonado no plasmídeo de expressão pFastBac 1, o qual possui o sítio promotor do gene da poliedrina. O subclone obtido foi analisado para certificação de sua orientação correta dentro do plasmídeo com as endonucleases de restrição BamH I e EcoR I. O subclone com a orientação correta teve seu DNA extraído e usado para a transposição dentro do bacmid (baculovírus recombinante e plasmídeo helper em célula competente DH10Bac ). As colônias com inserto gE foram selecionadas pelo fenótipo da colônia, a qual expressa cor branca quando clonada. As colônias brancas recombinantes tiveram seu DNA extraído e usado para a co-transfecção em células do inseto Trichoplusia ni (BTITn5B1- 4). O baculovírus gE-recombinante ao ser inoculado em cultivo celular, expressou a gE recombinante, comprovada pela técnica de PCR e Western blotting . O baculovírus gE-recombinante contendo apenas o gene da gE do VDA será utilizado para produção de antígeno e de anticorpos monoclonais, o que auxiliará no desenvolvimento de um teste de diagnóstico mais sensível, específico e mais seguro para uso em áreas livres do VDA