Bayreuth, Univ., Diss., 2004. / Computerdatei im Fernzugriff.

No description available.

A beta-glicosidase digestiva (Mr 50.000) de Spodoptera frugiperda (Sfgli50) possui em seu sítio ativo resíduos de aminoácido que interagem de forma não-covalente com o substrato. Essas interações não-covalentes garantem a especificidade da enzima em relação a seus substratos. Esse trabalho visa estudar o papel desses aminoácidos e suas interações com o substrato na especificidade da Sfgli50. Para isso, um modelo de previsão de especificidade baseado nas energias de interação entre diferentes resíduos de aminoácidos das posições 451 e 39 da Sfgli50 e as hidroxilas 4 e 6 do glicone do substrato foi elaborado e testado através da produção e caracterização de três duplos-mutantes (S451N39, S451E39 e A451E39) da Sfgli50. O modelo mostrou-se apropriado qualitativamente para previsão do comportamento da especificidade frente a glucosídeos e galactosídeos, mas totalmente inadequado para previsões de preferência frente a fucosídeos e galactosídeos. Estas conclusões indicaram que pressupostos iniciais do modelo, como independência das interações com o substrato e conservação estrutural do sítio ativo nos mutantes, estavam errados. Analisou-se também a contribuição de outros dois resíduos do subsítio do glicone para a especificidade da Sfgli50: H142 e N186, cujas energias de interação ainda não haviam sido determinadas. Experimentos de mutação sítio-dirigida foram realizados para introduzir resíduos de Alanina nas posições 142 e 186. Os mutantes H142A e N186A foram expressos em bactéria e em seguida parcialmente purificados. O mutante N186A apresentou baixa atividade catalítica, o que limitou sua caracterização. Por outro lado, a determinação de parâmetros cinéticos (Vmáx e Vmáx/Km) mostrou que, em contraste com a Sfgli50 selvagem, o mutante H142A é menos específico, principalmente no que diz respeito a fucosídeos. / The Mr 50,000 Spodoptera frugiperda beta-glycosidase (Sfgli50) has active site amino acid residues that interact non-covalently with the substrate. These non-covalent interactions determine the enzyme’s specificity towards its substrates. This work aims to study the role of these amino acids and its interactions with the substrate in the specificity of Sfgli50. A specificity prediction model was created for such purpose. This model is based on interaction energies between different amino acid residues in positions 451 and 39 of the enzyme and hydroxyls 4 and 6 of the substrate\'s glycone. The production and characterization of three double-mutants (S451N39, S451E39 and A451E39) were used to test the model, which proved to be qualitatively appropriate to predict the Sfgli50 specificity when comparing glucosides to galactosides. However, the model failed to predict the differences in specificity between fucosides and galactosides. These results indicate that the model\'s assumptions of independent interactions with the substrate and structural conservation of the active site in the mutants were wrong. Sfgli50 glycone residues H142 and N186, whose interaction energies had not been previously determined, were also investigated. Site-directed mutagenesis replaced the former residues in positions 142 and 186 for Alanine. The mutant proteins H142A and N186A were synthesized in bacteria and partially purified. Mutant N186A presented low catalytic activity, hindering its characterization. In contrast, mutant H142A is less specific than the wild-type Sfgli50. Kinetic parameters indicated that it is specially less specific to fucosides.

Thesis (doctoral)--Swedish University of Agricultural Sciences, 2003. / Appendix consists of reprints and manuscripts of five papers co-authored with others. Includes bibliographical references. Also partially available electronically via World Wide Web in PDF format; online version lacks appendix.

A beta-glicosidase digestiva (Mr 50.000) de Spodoptera frugiperda (Sfgli50) possui em seu sítio ativo resíduos de aminoácido que interagem de forma não-covalente com o substrato. Essas interações não-covalentes garantem a especificidade da enzima em relação a seus substratos. Esse trabalho visa estudar o papel desses aminoácidos e suas interações com o substrato na especificidade da Sfgli50. Para isso, um modelo de previsão de especificidade baseado nas energias de interação entre diferentes resíduos de aminoácidos das posições 451 e 39 da Sfgli50 e as hidroxilas 4 e 6 do glicone do substrato foi elaborado e testado através da produção e caracterização de três duplos-mutantes (S451N39, S451E39 e A451E39) da Sfgli50. O modelo mostrou-se apropriado qualitativamente para previsão do comportamento da especificidade frente a glucosídeos e galactosídeos, mas totalmente inadequado para previsões de preferência frente a fucosídeos e galactosídeos. Estas conclusões indicaram que pressupostos iniciais do modelo, como independência das interações com o substrato e conservação estrutural do sítio ativo nos mutantes, estavam errados. Analisou-se também a contribuição de outros dois resíduos do subsítio do glicone para a especificidade da Sfgli50: H142 e N186, cujas energias de interação ainda não haviam sido determinadas. Experimentos de mutação sítio-dirigida foram realizados para introduzir resíduos de Alanina nas posições 142 e 186. Os mutantes H142A e N186A foram expressos em bactéria e em seguida parcialmente purificados. O mutante N186A apresentou baixa atividade catalítica, o que limitou sua caracterização. Por outro lado, a determinação de parâmetros cinéticos (Vmáx e Vmáx/Km) mostrou que, em contraste com a Sfgli50 selvagem, o mutante H142A é menos específico, principalmente no que diz respeito a fucosídeos. / The Mr 50,000 Spodoptera frugiperda beta-glycosidase (Sfgli50) has active site amino acid residues that interact non-covalently with the substrate. These non-covalent interactions determine the enzyme’s specificity towards its substrates. This work aims to study the role of these amino acids and its interactions with the substrate in the specificity of Sfgli50. A specificity prediction model was created for such purpose. This model is based on interaction energies between different amino acid residues in positions 451 and 39 of the enzyme and hydroxyls 4 and 6 of the substrate\'s glycone. The production and characterization of three double-mutants (S451N39, S451E39 and A451E39) were used to test the model, which proved to be qualitatively appropriate to predict the Sfgli50 specificity when comparing glucosides to galactosides. However, the model failed to predict the differences in specificity between fucosides and galactosides. These results indicate that the model\'s assumptions of independent interactions with the substrate and structural conservation of the active site in the mutants were wrong. Sfgli50 glycone residues H142 and N186, whose interaction energies had not been previously determined, were also investigated. Site-directed mutagenesis replaced the former residues in positions 142 and 186 for Alanine. The mutant proteins H142A and N186A were synthesized in bacteria and partially purified. Mutant N186A presented low catalytic activity, hindering its characterization. In contrast, mutant H142A is less specific than the wild-type Sfgli50. Kinetic parameters indicated that it is specially less specific to fucosides.

We constructed a cDNA library with mRNA isolated from Sf9 cells infected with Spodoptera littoralis nucleopolyhedrovirus type B (SpliNPV-B) and identified the lef-3 gene from this library. Northern blot analysis showed that SpliNPV-B lef-3 mRNA was expressed as a 1.6 Kb transcript at 5 hours post infection (p.i.), reached high levels at 24 hours p.i., and remained highly expressed at 56 hours p.i.. Transcriptional mapping showed that lef-3 transcription started from two initiation sites (the distal and the proximal transcription initiation sites) located approximately 9 nucleotides apart. The sequences that modulate lef-3 expression were investigated by transient expression assays using a reporter gene under transcriptional control of the lef-3 promoter. Deletion analysis of the 5'-flanking region demonstrated that sequences up to 584 bases 5' of the distal transcription initiation site affected the level of reporter activity, indicating that this region contains transcription regulators. A region that was sufficient to direct basal level of promoter activity, the minimal promoter, was identified. This region encompasses the two transcription initiation sites, two TATA boxes, and a GATA motif. Mutations in the GATA motif resulted in substantial decrease in the level of reporter activity, suggesting that the GATA motif is an important element in the regulation of lef-3 gene expression. The sequence of a 2.6-kb region (mu 42.8-46.8) encompassing the lef-3 gene and flanking sequences was determined. Alignment of the predicted amino acid sequence of the LEF-3 polypeptide of SpliNPV-B with the putative sequences of AcMNPV and OpMNPV LEF-3 revealed low levels of sequence conservation (26% and 21% amino acid sequence identity, respectively). This low level of sequence conservation corroborates the view that, within the genus Nucleopolyhedrovirus, SpliNPV-B is distantly related to AcMNPV and OpMNPV. / Graduate

No description available.

This research investigated early transcriptional responses of the model legume, Medicago truncatula, to herbivory by caterpillars of the beet armyworm, Spodoptera exigua. Differentially expressed genes were identified by the differential display technique, cDNA-amplified fragment length polymorphism (cDNA-AFLP). To distinguish between caterpillar-specific responses and general wound responses, a subset of plants was mechanically damaged. Furthermore, to identify responses to salivary elicitors, plants were subject to herbivory by caterpillars with normal salivary secretions and those that had their spinneret, the appendage through which labial saliva is secreted, cauterized shut. Eighteen differentially expressed gene fragments, representing 16 genes, were identified. The expression pattern of 5 of these genes was analyzed by Northern analysis, confirming a caterpillar-specific reduction in transcripts encoding rubisco activase and glyceraldehyde-3-phosphate dehydrogenase. This research shows that plants are able to differentiate between caterpillar herbivory and mechanical damage and that transcriptional response are initiated within one hour after caterpillar infestation.

Baculoviruses are viruses of arthropods with large rod-shaped virions that contain supercoiled double-stranded DNA genomes. These viruses have been used as gene expression vectors and insect biological control agents, and have been studied as a virus model for the investigation of molecular mechanisms, such as apoptosis, gene expression, DNA replication, and virus-host interaction. Our current knowledge about baculovirus is largely based on the studies of the Autographa californica ucleopolyhedrovirus and the closely related species. In spite of the increasing interest of recombinant baculoviruses as gene expression and delivery vectors and bioinsecticides, the mechanisms of baculovirus DNA replication and virus-host interaction are still poorly understood. To take advantage of baculovirus diversity and their specific host-ranges, I studied the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV). Previous investigations indicated that SpliNPV possesses a unique host-range and genetic organization. In this dissertation, I studied the SpliNPV infection of an orthopteran cell line derived from the grasshopper, Melanopus sanguinipes, and provided evidence of viral DNA replication and production of viable virus progeny. I next investigated SpliNPV infection in five cell lines derived from three lepidopteran families: Sf9, CLS79 and Se1 cell lines from Spodoptera (Noctuidae), Ld652Y cells from Lymantria dispar (Lymantriidae), and Md210 cells from Malacosoma disstria (Lasiocampidae), which represented permissive (Sf9, CLS79, and Se1), semipermissive (Ld652Y), and non-permissive (Md210) cell lines. SpliNPV infection in permissive cell lines resulted in viral gene expression, DNA replication, and production of viable progeny. While the semi-permissive cell line displayed reduced and delayed transcription of viral genes and supported limited viral DNA replication, the non-permissive cell line displayed dramatically reduced viral transcription and abolished viral progeny. Transient expression assays using SpliNPV early- or very late-promoter reporters suggested that non-productive infection of SpliNPV in semi- or non-permissive cell lines was a consequence of limited viral specific transcription at the early phase of viral infection. Having documented the infection events in these cell lines, I investigated the mechanism of SpliNPV DNA replication. Using transient replication assays I have identified a non-hr origin of SpliNPV DNA replication. With limited sequence similarity to other NPV non-hr origins, the putative SpliNPV origin consists of sequence motifs found in other origins of virus DNA replication, such as imperfect palindromes, direct repeats, and transcription factor binding sites. Transient expression assays indicated that the putative non-hr origin represses the SpliNPV early gene, lef-3. Gel mobility shift analyses confirmed that nuclear proteins from both infected and uninfected cells bound with specificity to the putative origin. After identification and characterization of the cis-acting factor involved in viral DNA replication, I then identified a trans-acting factor involved in viral DNA replication. I have sequenced a 6.4 kb DNA from SpliNPV genome that contains an ORF encoding a predicated polypeptide of 998 amino acid sequences. Comparative sequence analyses demonstrated that the ORF encoded a DNA polymerase (dnapol) that consists of conserved exonuclease domains and DNA polymerase motifs found in other eukaryotic DNA viruses and in cellular DNA polymerases. The transcription initiation site of the 3 kb SpliNPV dnapol transcript was mapped to an NPV early promoter element, ACGT. The transcript terminated at the polyadenylation signal AATAAA. Using E. coli and baculovirus expression systems, I over-expressed a 110 kDa full-length SpliNPV DNA polymerase (DNAPOL) and a truncated 96 kDa protein, in which the amino terminal 80 amino acids were deleted. Enzymatic analyses demonstrated that the DNA polymerase and 3'- 5’exonuclease activities are intrinsic to the SpliNPV DNAPOL. Deletion of the 80 amino acid residues at the N-terminal of the DNAPOL did not affect DNA polymerase and exonuclease activities. Replication products from single-stranded M13 DNA revealed that SpliNPV DNAPOL possesses a proccessive activity. / Graduate

Appraisals of investment in pest control are complicated by the problems of predicting events in biological systems. In this study, an attempt is made to estimate the two necessary components of pest control investment appraisal, namely: the production function (decreases in crop losses with unit increases in pest control investment), and the pest damage function (relating crop damage to changing infestation variables), for attacks of the lepidopterous larvae of Spodoptera I1ttoralis (Boisd.), on Cypriot lucerne pastures. It is suggested that at present the best technique available to farmers for controlling S. littoralis infestations is the single application of one of three insecticides of proven efficacy. Consequently, the cost of successful pest control is represented by one value for a wide range of larval densities. The pest damage function is described as a dynamic relationship between a number of changing environmental and crop variab1es and is presented in the form of a computer simulation. This incorporates some of the existing empirical data on pest consumption and pest and crop interaction as well as much of the additional data collected by the author. The damage and production functions are compared, and estimates are made of the minimum larval density at various timings in the crop growth cycle, which is sufficient to cause losses equal to the treatment costs (the economic threshold of treatment). These estimates are offered as a basis for decision making on the economic control of S. littoralis in Cypriot lucerne fields.