Browsing by Subject "Molekulargenetik"

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Effekt der Überproduktion von Enzymen des Glucosestoffwechsels auf das Wachstum und die Alkoholbildung in der Hefe Saccharomyces cerevisiae
(2006) Emili, Markus; Heinisch, Jürgen
The wine-, beer- and baker's yeast Saccharomyces cerevisiae is the major source in world wide alcohol production. Regarding the research in bioethanol production, the work presented here was aimed to examine the effect of the in vivo overproduction of all enzymes contributing to the conversion of glucose to ethanol in the yeast Saccharomyces cerevisiae with the prospect of increasing ethanol formation. S. cerevisiae is probably the best studied eucaryotic organism with respect to both classical and molecular genetics. It turned out to be of great advantage that two different multi-copy-vectors could be employed in these studies. Each of them was used in the first part of the work to insert half of the set of genes intended for overexpression. The first genes were inserted by restriction and ligation and later on a combination of the PCR-technique, with which the genomic fragments of interest were amplified, and the efficient homologous recombination in vivo was used. With these methods, the gene encoding a hexose transporter (HXT1), all the genes encoding glycolytic enzymes (HXK2, PGI1, PFK1, PFK2, FBA1, TPI1, TDH1 bzw. TDH2, PGK1, GPM1, ENO2, PYK1), as well as the genes encoding enzymes needed for the conversion of pyruvate to ethanol (PDC1, ADH1), were cloned. Following the isolation from yeast, the plasmids were amplified in E. coli and characterized by restriction analysis. The measurement of specific enzyme activity in crude extract of yeast transformants with such plasmids showed a slight overproduction (factor 1,5 to 3,0) for all enzymes, except for glyceraldehyde-3-phosphate dehydrogenase. For HXT1, an increased mRNA level (factor 14 in contrast to the control) was taken as evidence for overproduction. In the enzymatic determinations a clear tendency showing a lower overproduction with an increasing number of genes on the plasmids was observed. These findings suggest a negative feedback on glycolytic flux regulation. The the growth rates obtained in the second part of the work also showed a clear reduction with increasing numbers of plasmid-encoded genes. Regarding the physiological parameters, no changes in the coefficients for glucose consumption and ethanol formation could be found in comparison to a wild-type control, and the yield remained basically unchanged as well. Interestingly, abolishing the ATP-inhibition of phosphofructokinase by expression of a mutant allele of PFK1, resulted in a faster growth of transformants with an otherwise isogenic background. This result indicates the physiological relevance of the allosteric regulation at this essential glycolytic step. A lack of enzyme activity in one of the glycolytic steps in deletion mutants normally leads to growth inhibition on hexoses. On this basis, the construction of a yeast strain was initiated with the objective to obtain stable multi-copy transformants simply by growing cells on different sugars as carbon sources. In detail, this was done by crossing a strain carrying a pgi1-deletion with a strain carrying a pyk1-deletion followed by sporulation and tetrad dissection. Preliminary data with intermediate strain constructs indicate a clear increase in plasmid stability after growing cells on complex media. From the results of this thesis, valuable insights into the regulation of the glycolytic flux in vivo can be deduced, which may serve as a basis for ongoing research on the improvement of ethanol formation by yeast.
Molecular evidence of intraclonal variation and implications for adaptational traits of grape phylloxera populations (Daktulosphaira vitifoliae, Fitch)
(2007) Vorwerk, Sonja; Blaich, Rolf
Grape phylloxera (Daktulosphaira vitifoliae Fitch; Homoptera: Phylloxeridae) is an economical important insect pest of grapevine (Vitis spp.) worldwide. The insect was introduced with contaminated plant material from North America in the 1850s and spread rapidly across all European viticultural regions. In the 19th century, nearly three-forths of the ungrafted and highly susceptible European grape species were destroyed by the insect pest. European viticulture did not recover until the development of grafting, combining European Vitis vinifera varieties with resistant rootstocks, bred from American Vitis species. Grape phylloxera is still present in viticulture. Today, grape phylloxera populations mainly persist in abandoned vineyards and rootstock nurseries. Grape phylloxera populations seem to be variable in terms of genotypic composition and host adaptability. The lifecycle described by Fitch (1854) and others in the 19th century does not seem to match actual conditions anymore. This thesis aimed at redefining the genetic structure of European grape phylloxera populations by employing genetic markers. It was shown, that the insect has turned away from its classical holocycle and now mainly reproduces asexually, as already demonstrated for Australian grape phylloxera populations. Despite asexual reproduction, all examined populations revealed a high grade of genotypic diversity. The reports on the emergence of new and more aggressive strains raised the question, how a population composed of asexually reproducing organisms would change and adapt to such an extent. Using a multilocus marker system, eight single founder lineages were genetically monitored over at least 15 generations. All lineages revealed a high grade of intraclonal variation. Sequencing of polymorphic fragments showed, that the genetic variation was not due to contaminating plant or bacterial DNA, but was due to variation within the insect genome. Furthermore, mutations occured already in early generations and were not observed to accumulate in later generations. Mutations were rather generated constantly and only few mutation specific markers were identified to be stable over all following generations. The here documentated genetic variation reveals the great adaptational potential of this insect pest. The adaptability of single founder lineages was further assessed by measuring physiological parameters in single isolation chambers in the greenhouse. Parameters as the number of surviving individuals per generation, the number of eggs or the number of ovarioles per generation exposed differences in performance among the lineages and also within the lineages a high grade of intraclonal variation. A direct correlation of specific multilocus markers and particularly adapted individuals or lineages was not possible in this assay. Two markers, though, were observed to occure in several lineages which performed well on the new host plant. These markers may be a first step to the development of adaptation-related markers and need to be tested on further populations and host plants. When analysing intraclonal variation, the question of putative contaminating factors within the system arises. Symbiotic bacteria occuring in nearly all aphid species certainly are the first to be suspected as a source of genetic variation among single individuals tested. Endosymbiotic bacteria, as Buchnera aphidicola in other aphid species, influencing nutritional condition and fitness of the insect population, were not identified in D. vitifoliae. A bacterium, closely related to Pantoea agglomerans, however, was identified in several grape phylloxera populations, using universal 16S rDNA primers and later specifically developed markers, which were also employed for in situ hybridization. The bacterium was localized in the salivary pump of D. vitifoliae. PCR analysis of in vitro reared populations revealed that the bacterium is present in root- and leaf-feeding parthenogenetic populations of grape phylloxera and, moreover, seems to be transmitted from generation to generation. In other insect species, this bacterium has been demonstrated to produce antifungal and antibacterial substances, which were also found in first in vitro tests with grape phylloxera associated bacteria. The insect may benefit from the antagonistic potential of these bacteria. P. agglomerans may be a further participant in the certainly complex interaction of grape phylloxera and grapevine. This thesis represents a broad approach to elucidate the development of grape phylloxera populations in Europe. Using new molecular marker systems, it has become possible to gain more information on the genetic structure of the insect and its adaptational potential. The predominant clonal reproduction mode of the insect confronts grapevine breeders and pest management with the task to continously develop new resistant rootstocks and to keep up with new pest management systems.