Transcriptome analysis of a respiratory Saccharomyces cerevisiae strain suggests the expression of its phenotype is glucose insensitive and predominantly controlled by Hap4, Cat8 and Mig1

BACKGROUND: We previously described the first respiratory Saccharomyces cerevisiae strain, KOY.TM6*P, by integrating the gene encoding a chimeric hexose transporter, Tm6*, into the genome of an hxt null yeast. Subsequently we transferred this respiratory phenotype in the presence of up to 50 g/L glucose to a yeast strain, V5 hxt1-7Delta, in which only HXT1-7 had been deleted. In this study, we compared the transcriptome of the resultant strain, V5.TM6*P, with that of its wild-type parent, V5, at different glucose concentrations. RESULTS: cDNA array analyses revealed that alterations in gene expression that occur when transitioning from a respiro-fermentative (V5) to a respiratory (V5.TM6*P) strain, are very similar to those in cells undergoing a diauxic shift. We also undertook an analysis of transcription factor binding sites in our dataset by examining previously-published biological data for Hap4 (in complex with Hap2, 3, 5), Cat8 and Mig1, and used this in combination with verified binding consensus sequences to identify genes likely to be regulated by one or more of these. Of the induced genes in our dataset, 77% had binding sites for the Hap complex, with 72% having at least two. In addition, 13% were found to have a binding site for Cat8 and 21% had a binding site for Mig1. Unexpectedly, both the up- and down-regulation of many of the genes in our dataset had a clear glucose dependence in the parent V5 strain that was not present in V5.TM6*P. This indicates that the relief of glucose repression is already operable at much higher glucose concentrations than is widely accepted and suggests that glucose sensing might occur inside the cell. CONCLUSION: Our dataset gives a remarkably complete view of the involvement of genes in the TCA cycle, glyoxylate cycle and respiratory chain in the expression of the phenotype of V5.TM6*P. Furthermore, 88% of the transcriptional response of the induced genes in our dataset can be related to the potential activities of just three proteins: Hap4, Cat8 and Mig1. Overall, our data support genetic remodelling in V5.TM6*P consistent with a respiratory metabolism which is insensitive to external glucose concentrations.

Proteolytic activity amongst selected Saprolegnia species

Saprolegia ssp. effectively utilized the protein casein as a sole source of carbon, nitrogen and sulphur, indicating considerable proteolytic activity. In the presence of a more simple carbon source such as glucose, which was readily assimilated, catabolite repression was not observed and casein exploitation was enhanced. Free proteinase activity was not detected by a number of methods, irrespective of culture conditions. However, clearing by mycelia of skimmed milk agar or agar amended with bacteria demonstrated a close association between proteinases and hyphae, suggestive of natural immobilization of proteinases. Casein breakdown was accompanied by release of individual amino acids and ammonia. The latter, indicative of amino acid assimilation and metabolism, was also associated with an increase in pH of culture medium. Single amino acids did not support growth of Saprolegnia but in combination with other amino acids, methionine encouraged greatest biomass production. Certain groupings of amino acids affected growth in a manner which departed from that expected, as assessed by multifactorial analysis of variance, and either enhanced or reduced growth.

Down-regulation of central glucocorticoid receptor expression using antisense oligonucleotide technology

Endogenous glucocorticoids and serotonin have been implicated in the pathophysiology of depression, anxiety and schizophrenia. This thesis investigates the potential of downregulating expression of central Type II glucocorticoid receptors (GR) both in vitro and in vivo, with empirically-designed antisense oligodeoxynucleotides (ODN), to characterise GR modulation of 5-HT2A receptor expression using quantitative RT-PCR, Western blot analysis and radioligand binding. The functional consequence of GR downregulation is also determined by measuring 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane hydrochloride (DOI) mediated 5-HT2A receptor specific headshakes. Using a library of random antisense ODN probes, RNAse H accessibility mapping of T7-primed, in vitro transcribed GR mRNA revealed several potential cleavage sites and identified an optimally effect GR antisense ODN sequence of 21-mer length (GRAS5). In vitro efficacy studies using rat C6 glioma cells showed a 56% downregulation in GR mRNA levels and 80% downregulation in GR protein levels. In the same cells a 29% upregulation in 5-HT2A mRNA levels and 32% upregulation in 5-HT2A protein levels was revealed. This confirmed the optimal nature of the GRAS5 sequence to produce marked inhibition of GR gene expression, and also revealed GR modulation of the 50-HT2A receptor subtype in C6 glioma cells to be a tonic repression of receptor expression. The distribution of a fluorescently-labelled GRAS5 ODN was detected in diverse areas of the rat brain after single ICV administration, although this fluorescence signal was not sustained over a period of 5 days. However, fluorescently-labelled GRAS5 ODN, when formulated in polymer microspheres, showed diverse distribution in the brain which was maintained for 5 days following a single ICV administration. This produced no apparent neurotoxic effects on rat behaviour and hypothalamic-pituitary-adrenal (HPA) axis homeostasis. Furthermore, a single polymer microsphere injection ICV proved to be an effective means of delivering antisense ODNs and this was adopted for the in vivo efficacy studies. In vivo characterisation of GRAS5 revealed marked downregulation of GR mRNA in rat brain regions such as the frontal cortex (26%), hippocampus (35%), and hypothalamus (39%). Downregulation of GR protein was also revealed in frontal cortex (67%), hippocampus (76%), and hypothalamus (80%). In the same animals upregulation of 5-HT2A mRNA levels was shown in frontal cortex (13%), hippocampus (7%), and hypothalamus (5%) while upregulation in 5-HT2A protein levels was shown in frontal cortex (21 %). This upregulation in 5-HT2A receptor density as a result of antisense-mediated inhibition of GR was further confirmed by a 55% increase in DOl-mediated 5-HT2A receptor specific headshakes. These results demonstrate that GR is involved in tonic inhibitory regulation of 5-HT2A receptor expression and function in vivo, thus providing the potential to control 5-HT2A-linked disorders through corticosteroid manipulation. These experiments have therefore established an antisense approach which can be used to investigate pharmacological characteristics of receptors.

The effects of the environment on the stability and expression of genetically engineered plasmids

The lac promoter is widely used in plasmid expression systems, even though it is prone to catabolite repression. As a consequence glycerol is often used as an alternative carbon source. Three plasmids containing various sizes of the staphylococcal protein A (SPA) gene, which are under the control of the lac promoter were investigated in continuous culture, to evaluate the effects of nutrient limitations on their stability and expression. The fears of catabolite repression were dispelled as a low expression plasmid (pPA16) produced a greater amount of truncated SPA under glucose limiting conditions (11 ug mg-1 cell protein) when compared to that using glycerol (8 ug mg-1 cell protein). Segregational instability was also observed under glycerol limiting conditions at all the dilution rates investigated. Whereas pPA16 was relatively stable under glucose limiting conditions, with SPA production being continuous. Experiments using excess glycerol with limited ammonium increased the stability of pPA16, (when compared to limited glycerol) with expression of SPA being continuous but reduced (6 ug mg-1 cell protein). With excess glucose and limited ammonium the copy numbers remained high but expression of SPA paralled that produced under glucose limiting conditions. This might indicate that the higher levels of glucose are reducing expression (catabolite repression) or that the low level of ammonium is affecting protein production. A high expression plasmid (pPA31) produced nearly 100 ug full length SPA mg-1 cell protein, while another high expression plasmid (pPA34) producing truncated SPA proved to be very unstable. An ELISA was developed to detect the SPA produced by these experiments, which could be adapted for western blotting or immunogold probing using electron microscopy. SPA was localised in electron lucent areas present in the periplasmic space of the E. coli host harbouring pPA16. While in the same host containing pPA31, SPA was localised not only in electron lucent areas but also around the whole of the outer-membrane.

Finance and entrepreneurial entry

This paper investigates the determinants of both the volume of finance and the sources of finance of business start-ups in 42 countries. Using the Global Entrepreneurship Monitor (GEM) surveys for 1998-2003, we jointly examine how the institutional business environment and individual characteristics of entrepreneurs affect the financing of entrepreneurs. We find that the property rights system is the most significant determinant of both the total volume of finance and of the use of external finance for the individual start-up project. In addition, our findings suggest that that the supply of informal finance is associated with the higher share of external sources in start-up finance, whereas the size of the formal financial sector appears to play a more important role for the volume of entrepreneurial self-finance. Our results suggest that the use of external finance by start-ups correlates with the extent of financial restrictions in a country in a non-monotonic way. We find that some financial restrictions may enhance the use of external funds by entrepreneurs, yet the sign of this effect reverses and the coefficient becomes stronger for the high level of financial repression by the government. Finally, our findings imply that the characteristics of the start-up finance are also affected by various individual characteristics of entrepreneurs, their growth intentions and ownership structure.

Diesseits und jenseits der Oder:Judith Hermanns literarische Auseinandersetzung mit Theodor Fontane in Sommerhaus, später

Judith Hermann's works have attracted considerable criticism for their supposedly slight portrayal of passively drifting characters and for their alleged failure to engage with the socio-political realities of contemporary life in the Berlin Republic. Only very recently have scholars paid attention to the hidden concern with memory expressed in her books, and have set out to examine their intertextual depth. This paper explores these previously neglected historical references in Summerhouse, later and analyses the book's intricate intertextual allusions with specific reference to Theodor Fontane's works. It examines how the tentative existence, which Hermann's characters experience, is the product of a hesitant and fruitless confrontation with questions of German history and nationhood. Using pervasive water imagery, Hermann shows present-day Germany as a continually contested territory with a fluid identity shaped by an abundance of conflicting narratives. In this context, the allusions to Fontane as a representative of the Wilhelminian period serve as references to a continuing German tradition of repression and marginalisation. At the same time, Hermann recognises Fontane's ambivalent political stance combining elements of social criticism with a general endorsement of social order. Ultimately, the seemingly indifferent attitude of Hermann's characters and the elegiac style used to portray them, emerge as a distancing mechanism that functions as a postmodern variant of Fontane's irony and is shaped by a similar sense of skepticism towards developments in German society and national history. © 2012 Springer Science+Business Media B.V.

Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level

Background: The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale. Results: We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol. Conclusions: P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to different growth conditions in P. pastoris is transcriptional; translational regulation was not transcript-specific. The high proportion of mRNAs associated with polysomes in methanol-grown cells is a major finding of this study; it reveals that high productivity during methanol induction is directly linked to the growth condition and not only to promoter strength.

REST is a hypoxia-responsive transcriptional repressor

Cellular exposure to hypoxia results in altered gene expression in a range of physiologic and pathophysiologic states. Discrete cohorts of genes can be either up- or down-regulated in response to hypoxia. While the Hypoxia-Inducible Factor (HIF) is the primary driver of hypoxia-induced adaptive gene expression, less is known about the signalling mechanisms regulating hypoxiadependent gene repression. Using RNA-seq, we demonstrate that equivalent numbers of genes are induced and repressed in human embryonic kidney (HEK293) cells. We demonstrate that nuclear localization of the Repressor Element 1-Silencing Transcription factor (REST) is induced in hypoxia and that REST is responsible for regulating approximately 20% of the hypoxia-repressed genes. Using chromatin immunoprecipitation assays we demonstrate that REST-dependent gene repression is at least in part mediated by direct binding to the promoters of target genes. Based on these data, we propose that REST is a key mediator of gene repression in hypoxia.