Anorectal malformations

Abstract The research described in this thesis was performed with the aim to evaluate and improve multidisciplinary treatment of anorectal malformation patients. An overview of current literature on treatment of anorectal malformations is given in the Preface section, which also includes an overview of this thesis. The results of the research are presented in two parts: Part 1 focuses on postnatal care and contains retrospective studies, while Part 2 focuses on long-term outcome from childhood into adulthood, and presents a cohort study and prospective studies. Then, the General discussion, Recommendations, and Summaries form Part 3, and Appendices are provided in Part 4

A vector for Aspergillus transformation conferring phleomycin resistance.

Recently, transformation of Aspergillus species with vector pAN7-1, conferring resistance to hygromycin B was reported (Punt et al. 1987 Gene 56:117-124)

Linkage mapping of the gpdA gene of Aspergillus nidulans

In the last few years many genes of several Aspergillus species have been cloned and sequenced. For many of these genes mutant alleles and genetic linkage data are also available. However, for those genes for which no mutant alleles have been isolated, genetic mapping was not possible. Here we report linkage mapping of the glyceraldehyde-3- phosphate dehydrogenase gene (gpdA) of A. nidulans for which no mutant alleles have been isolated. The method used is applicable to all other cloned genes

The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and V(HH)) by these lower eukaryotes and the possible applications of these proteins. Also the coupling of fragments to relevant enzymes or other components will be discussed. As an example of the fusion protein strategy, the 'magic bullet' approach for industrial applications, will be highlighted

GTP-binding protein Era: a novel gene target for biofuel production

Background: Biodiesel production using cyanobacteria is a promising alternative to fossil fuels. In this study we created a transposon library of Synechococcus elongatus PCC 7942 in order to identify novel gene targets for enhanced fatty acid and hydrocarbon production. The transposon library was subsequently screened for desirable traits using macro- and microscopic observations as well as staining with the lipophilic dye Nile Red. Results: Based on the screening results, we selected a single mutant, which has an insertion in the gene encoding for the GTP-binding protein Era. We subsequently verified the phenotype-genotype relation by overexpression, reintroducing and complementing the mutation. Overexpression of era caused a reduction in the cell size in the late exponential phase of growth and an increase in the total amount of intracellular fatty acids. Reintroduction of the inactivated transposon caused a significant increase in the cellular length as well as changes in the amounts of individual hydrocarbons and fatty acids. Ectopic complementation of this mutation fully restored the hydrocarbon production profile to that of wild-type and partially restored the fatty acid production. Moreover, the cellular size was significantly smaller than that of the inactivated transposon mutant. Conclusions: The GTP-binding protein Era has never been studied in cyanobacteria and proved to be an essential gene for S. elongatus PCC 7942. We also found that this protein is important for hydrocarbon and fatty acid metabolism as well as determination of the cell size in PCC 7942. Our results suggest that the GTP-binding protein Era can be used as a novel target for further improvement of biofuel precursors production

Transcriptomic comparison of Aspergillus niger growing on two different sugars reveals coordinated regulation of the secretory pathway

<p>Abstract</p> <p>Background</p> <p>The filamentous fungus, <it>Aspergillus niger</it>, responds to nutrient availability by modulating secretion of various substrate degrading hydrolases. This ability has made it an important organism in industrial production of secreted glycoproteins. The recent publication of the <it>A. niger </it>genome sequence and availability of microarrays allow high resolution studies of transcriptional regulation of basal cellular processes, like those of glycoprotein synthesis and secretion. It is known that the activities of certain secretory pathway enzymes involved <it>N</it>-glycosylation are elevated in response to carbon source induced secretion of the glycoprotein glucoamylase. We have investigated whether carbon source dependent enhancement of protein secretion can lead to upregulation of secretory pathway elements extending beyond those involved in <it>N</it>-glycosylation.</p> <p>Results</p> <p>This study compares the physiology and transcriptome of <it>A. niger </it>growing at the same specific growth rate (0.16 h^-1) on xylose or maltose in carbon-limited chemostat cultures. Transcription profiles were obtained using Affymetrix GeneChip analysis of six replicate cultures for each of the two growth-limiting carbon sources. The production rate of extracellular proteins per gram dry mycelium was about three times higher on maltose compared to xylose. The defined culture conditions resulted in high reproducibility, discriminating even low-fold differences in transcription, which is characteristic of genes encoding basal cellular functions. This included elements in the secretory pathway and central metabolic pathways. Increased protein secretion on maltose was accompanied by induced transcription of > 90 genes related to protein secretion. The upregulated genes encode key elements in protein translocation to the endoplasmic reticulum (ER), folding, <it>N</it>-glycosylation, quality control, and vesicle packaging and transport between ER and Golgi. The induction effect of maltose resembles the unfolded protein response (UPR), which results from ER-stress and has previously been defined by treatment with chemicals interfering with folding of glycoproteins or by expression of heterologous proteins.</p> <p>Conclusion</p> <p>We show that upregulation of secretory pathway genes also occurs in conditions inducing secretion of endogenous glycoproteins – representing a more normal physiological state. Transcriptional regulation of protein synthesis and secretory pathway genes may thus reflect a general mechanism for modulation of secretion capacity in response to the conditional need for extracellular enzymes.</p

Agrobacterium-mediated transformation as a tool for functional genomics in fungi

Plant sciencesMicrobial Biotechnolog