Yeast Los1p has properties of an exportin-like nucleocytoplasmic transport factor for tRNA.

Saccharomyces cerevisiae Los1p, which is genetically linked to the nuclear pore protein Nsp1p and several tRNA biogenesis factors, was recently grouped into the family of importin/karyopherin-beta-like proteins on the basis of its sequence similarity. In a two-hybrid screen, we identified Nup2p as a nucleoporin interacting with Los1p. Subsequent purification of Los1p from yeast demonstrates its physical association not only with Nup2p but also with Nsp1p. By the use of the Gsp1p-G21V mutant, Los1p was shown to preferentially bind to the GTP-bound form of yeast Ran. Furthermore, overexpression of full-length or N-terminally truncated Los1p was shown to have dominant-negative effects on cell growth and different nuclear export pathways. Finally, Los1p could interact with Gsp1p-GTP, but only in the presence of tRNA, as revealed in an indirect in vitro binding assay. These data confirm the homology between Los1p and the recently identified human exportin for tRNA and reinforce the possibility of a role for Los1p in nuclear export of tRNA in yeast.

Direct simulation of heterogeneous diffusion and inversion procedure applied to an out-diffusion experiment. Test case of Palmottu granite.

An out-diffusion laboratory experiment using a non-reactive tracer was fitted using the Time Domain Diffusion (TDD) method. This rapid particle tracking method allows simulation of the heterogeneous diffusion based on pore-scale images and local values of diffusivities. The superimposed porosity and mineral 2D maps act as computation grids to condition diffusion pathways. We focused on a Palmottu granite sample, in which the connected pore space has a composite microstructure with cracks linking microporous minerals and is above the percolation threshold. Three main results were achieved: (i) When compared to the fitting obtained with one coefficient (best mean square residual R = 1.6 x 10(-2)), diffusion is shown to be suitably characterised with two coefficients related to cracks and microporous minerals (best R = 6.5 x 10(-4)), (ii) rather than imposing a local apparent diffusion coefficient D(a) independent of the local porosity Phi, a best fit is obtained by applying Archie's relationship D(a) = D(0) x G with G = Phi(m) to each pixel of the calculation grids (G is the geometry factor, D(0) is the diffusion coefficient in free fluid, and m is Archie's exponent), and (iii) the order of magnitude of the fitted diffusion coefficient or Archie's exponents (m=0 for microcracks and m=1.82 for microporous minerals) is physically realistic.

Cloning and characterization of the Schizosaccharomyces pombe tRNA:pseudouridine synthase Pus1p.

Saccharomyces cerevisiae cells that carry deletions in both the LOS1 (a tRNA export receptor) and the PUS1 (a tRNA:pseudouridine synthase) genes exhibit a thermosensitive growth defect. A Schizosaccharomyces pombe gene, named spPUS1, was cloned from a cDNA library by complementation of this conditional lethal phenotype. The corresponding protein, spPus1p, shows sequence similarity to S. cerevisiae and murine Pus1p as well as other known members of the pseudouridine synthase family. Accordingly, recombinant spPus1p can catalyze in vitro the formation of pseudouridines at positions 27, 28, 34, 35 and 36 of yeast tRNA transcripts. The sequence and functional conservation of the Pus1p proteins in fungi and mammalian species and their notable absence from prokaryotes suggest that this family of pseudouridine synthases is required for a eukaryote-specific step of tRNA biogenesis, such as nuclear export.

Effect of growth rate on stability and gene expression of recombinant plasmids during continuous and high cell density cultivation of Escherichia coli TG1.

Continuous and fed-batch cultures of recombinant Escherichia coli TG1 were carried out in order to study plasmid stability and recombinant product formation at different specific growth rates. The aprotinin::beta-galactosidase gene (Ap::lacZ) was placed under the control of two different promoter/repressor systems, the PLac/lacI (pPLac8) and the lambda PL/cIts857 (pPL6) system. The chemically (0.5 mM IPTG) induced gene expression exhibited higher product activity and plasmid stability than the thermally (40 degrees C) induced expression. In fed-batch cultivations with the more stable E. coli TG1(pPLac8) a special feeding strategy allowed bacterial growth with a constant growth rate mu for several hours up to high cell densities. The cloned gene product activity was noticeably effected by the specific growth rate and the cell density at the moment of induction. In particular, the enzyme activities passed a pronounced maximum value in dependence of the set growth rate. The results indicate that fed-batch cultivation strategies are well suited to produce recombinant gene products.

Simple fed-batch technique for high cell density cultivation of Escherichia coli.

A simple fed-batch process for high cell density cultivation of Escherichia coli TG1 was developed. A pre-determined feeding strategy was chosen to maintain carbon-limited growth using a defined medium. Feeding was carried out to increase the cell mass concentration exponentially in the bioreactor controlling biomass accumulation at growth rates which do not cause the formation of acetic acid (mu < mu crit). Cell concentrations of 128 and 148 g per 1 dry cell weight (g l-1 DCW) were obtained using glucose or glycerol as carbon source, respectively.

Fungal morphology in submerged cultures and its relation to glucose oxidase excretion by recombinant Aspergillus niger.

The effect of culture conditions such as medium composition and shear stress on the fungal pellet morphology in shake-flask cultures and its relation to glucose oxidase (GOD) excretion by recombinant Aspergillus niger NRRL 3 (GOD 3-18) was investigated. It was shown that culture conditions resulting in the formation of smaller fungal pellets with an increased mycelial density result in higher yields of exocellular GOD. The pellets obtained in shake-flask cultures showed distinct layers of mycelial density with only the thin outer layer consisting of a dense mycelial network. The performance of the recombinant strain and the process of pellet formation was also analyzed during batch cultivation in a stirred-tank bioreactor. It was shown that the process of pellet formation occurred in two steps: (1) aggregation of free spores to spore clusters with subsequent germination and formation of small aggregates surrounded by a loose hyphal network, and (2) aggregation of the primary aggregates to the final full-size pellets. The fungal pellets formed during bioreactor cultivation were smaller, did not show large differences in mycelial density, and were more efficient with respect to the production of exocellular GOD. The decreasing pellet size also correlated with an increased mycelial density, indicating an improvement of the transport of nutrients to the inner parts of the pellet.

GpdA-promoter-controlled production of glucose oxidase by recombinant Aspergillus niger using nonglucose carbon sources.

The gpdA-promoter-controlled exocellular production of glucose oxidase (GOD) by recombinant Aspergillus niger NRRL-3 (GOD3-18) during growth on glucose and nonglucose carbon sources was investigated. Screening of various carbon substrates in shake-flask cultures revealed that exocellular GOD activities were not only obtained on glucose but also during growth on mannose, fructose, and xylose. The performance of A. niger NRRL-3 (GOD3-18) using glucose, fructose, or xylose as carbon substrate was compared in more detail in bioreactor cultures. These studies revealed that gpdA-promoter-controlled GOD synthesis was strictly coupled to cell growth. The gpdA-promoter was most active during growth on glucose. However, the unfavorable rapid GOD-catalyzed transformation of glucose into gluconic acid, a carbon source not supporting further cell growth and GOD production, resulted in low biomass yields and, therefore, reduced the advantageous properties of glucose. The total (endo- and exocellular) specific GOD activities were lowest when growth occurred on fructose (only a third of the activity that was obtained on glucose), whereas utilization of xylose resulted in total specific GOD activities nearly as high as reached during growth on glucose. Also, the portion of GOD excreted into the culture fluid reached similar high levels (approximately equal to 90%) by using either glucose or xylose as substrate, whereas growth on fructose resulted in a more pelleted morphology with more than half the total GOD activity retained in the fungal biomass. Finally, growth on xylose resulted in the highest biomass yield and, consequently, the highest total volumetric GOD activity. These results show that xylose is the most favorable carbon substrate for gpdA-promoter-controlled production of exocellular GOD.

Effect of moisture content on emanation at different grain size fractions - a pilot study on granitic esker sand sample.

It is known that in soils and sediments moisture adsorbed on particle surfaces and in the pore system significantly affects the behaviour of recoiling radon ((222)Rn) atoms after decay of parent (226)Ra, leading to increased (222)Rn emanation. As a first step in an effort to characterize the (222)Rn source term in mineralised sediments in the present study, complementing previous studies in the area, granitic esker sand samples were collected in order to test how moisture content affects (222)Rn emanation at different grain size fractions. Emanation fractions measured for natural samples were compared with theoretical calculations. Six different grain size fractions were studied at 0%, 5% and 10% moisture contents relative to the mass of solids. In a further study necessary complementary information on the chemical and structural distribution of (226)Ra was gained by selective leaching experiments. The results showed that (226)Ra concentration increases from 50 Bq/kg at grain size 1-2 mm to 200 Bq/kg at grain size <0.063 mm. Respectively, the emanation factor increased from 0.12 to 0.30 at 5% moisture content. Both emanation factor and radium concentration increased significantly when grain size was below 0.125-0.250 mm. Above this fraction, the emanation fraction was approximately constant, 0.13 at 5% moisture content. In most of the grain size fractions, emanation reaches its maximum at 5% moisture content, being twice as high as in a dry sample. For the small particles (<0.063 mm) the (226)Ra distribution is rather complex and depends on the mineral composition compared to larger particles wherein emanation from the internal pore system and the adjacent matrix is dominating over the contribution from external surface.

Kinetics of glucose oxidase excretion by recombinant Aspergillus niger.

The kinetics of glucose oxidase (GOD) excretion by recombinant Aspergillus niger NRRL-3 (GOD3-18) were investigated using enzymatic activity measurements as well as gel electrophoresis techniques. The majority of GOD was produced during rapid growth in the first phase of the cultivation. The high excretion rate during this phase did not prevent the endocellular accumulation of GOD up to 40% of the total soluble cell protein demonstrating that the production rate exceeded the excretion rate of the enzyme into the culture medium. During the second phase of the cultivation, excretion of GOD occurred at a slower rate, although the majority of GOD produced during the first phase was excreted during the second phase of the cultivation. At the end, about 90% of the total GOD produced was recovered from the culture medium. Two-dimensional gel electrophoresis provided evidence that endo- and exocellular GOD were indistinguishable, revealing identical posttranslational modifications (e.g., signal sequence cleavage, glycosylation pattern). The results demonstrate that the initial steps of the secretory pathway are fast and that the excretion of the enzyme into the culture fluid was most likely delayed due to retention by the cell wall.

[The Helkimo index for assessing treatment results after mandibular fractures]. / Der Helkimo-Index als Beurteilungsmöglichkeit der Behandlungsergebnisse von Unterkieferfrakturen.

Based on the Helkimo index we assessed the success of conservative and conservative-surgical forms of treatment for mandibular fractures in a total of 166 patients. The control group consisted of 50 probands. The Helkimo dysfunction index, which is based on various criteria, showed dysfunction in 81.9% of the patients with only slight functional impairment in 57.2% of these cases. 18.1% of the patients were clinically symptom-free. A comparison with the probands revealed no major differences in dysfunction between the two groups. Significant differences were observed only in the case of 2 isolated criteria (maximum mandibular protrusion, muscle pain). Severe dysfunction, however, was more common in the fracture patients. Based on the Helkimo occlusion index disorder were noted in 91.7% of the patients, while severe occlusion disorder were observed particularly in patients with combined mandibular body and condyle fractures (40.0%). In the control group a more favorable occlusion index was found to be due mainly to the greater number of present and/or occluding teeth. The Helkimo index, particularly the dysfunctionindex, is a very useful instrument for assessing the success of treatment measures in mandibular fracture cases. It should be more commonly used to improve the possibilites of objective comparisons between patients from different hospitals.

[The radioecology of the grapevine. 1. The transfer of nuclear weapons fallout from the soil into wine]. / Zur Radioökologie der Weinrebe. Teil 1. Transfer von Kernwaffenfallout vom Boden in den Wein.

In a field investigation (1983-1985) comprising eight places of the most important viticultural regions in the Federal Republic of Germany, the contents of the radionuclides tritium (3H), carbon-14 (14C), strontium-90 (90Sr), and cesium-137 (137Cs) in air, soils, leaves of the vine, grapes and wine were measured and site-specific transfer factors were calculated. Data concerning soil parameters, climatic conditions, cultivation and vinification were collected. The tritium content of all samples was 10 Bq/l water of combustion, independent of location and year. The specific activity of 14C in the atmosphere and in biological material was 0.22 Bq/g carbon, independent of site and year. 90Sr contents of soils fluctuated between 0.7 and 3.5 Bq/kg dry matter. The mean content of leaves was 2 Bq/kg fresh material, of grapes 0.035 Bq/kg and of wine 0.008 Bq/l. 137Cs content of soils fluctuated between 1.3 and 7.9 Bq/kg dry matter. The mean content of leaves was 0.098 Bq/kg fresh material, of grapes 0.021 Bq/kg and of wine 0.0085 Bq/l. A relation between transfer of radionuclides and soil parameters and between the contents of grapes and wine was not recognizable. While cultivar-specific differences were not observed in grapes, red wines contained somewhat more 137Cs than white wines. Transfer factors soil grapes were 0.027 for 90Sr and 0.0057 for 137Cs. Site-specific influences such as soil parameters, climate, cultivation, vinification and differences between years led to a relatively small fluctuation of values. An influence of the nuclear power station Neckarwestheim has not been found in any of the radionuclides.

Entry of Escherichia coli into stationary phase is indicated by endogenous and exogenous accumulation of nucleobases.

Endogenous and exogenous accumulation of nucleobases was observed when Escherichia coli entered the stationary phase. The onset of the stationary phase was accompanied by excretion of uracil and xanthine. Except for uracil and xanthine, other nucleobases (except for minor amounts of hypoxanthine), nucleosides, and nucleotides (except for cyclic AMP) were not detected in significant amounts in the culture medium. In addition to exogenous accumulation of nucleobases, stationary-phase cells increased the endogenous concentrations of free nucleobases. In contrast to extracellular nucleobases, hypoxanthine was the dominating intracellular nucleobase and xanthine was present only in minor concentrations inside the cells. Excretion of nucleobases was always connected to declining growth rates. It was observed in response to entry into the stationary phase independent of the initial cause of the cessation of cell growth (e.g., starvation for essential nutrients). In addition, transient accumulation of exogenous nucleobases was observed during perturbations of balanced growth conditions such as energy source downshifts. The nucleobases uracil and xanthine are the final breakdown products of pyrimidine (uracil and cytosine) and purine (adenine and guanine) bases, respectively. Hypoxanthine is the primary degradation product of adenine, which is further oxidized to xanthine. The endogenous and exogenous accumulation of these nucleobases in response to entry into the stationary phase is attributed to degradation of rRNA.

Translational coupling varying in efficiency between different pairs of genes in the central region of the atp operon of Escherichia coli.

A series of atp::lacZ fusions has been constructed for use in a study of translational coupling in the central region of the Escherichia coli atp operon. Five genes, atpE, atpF, atpH, atpA and atpG, were shown to be translationally coupled to various degrees of tightness. A new lac promoter vector, compatible with the atp::lacZ fusion vectors, was used to express individual atp genes in the same hosts as the fusion genes. The H(+)-ATPase subunits thus synthesized exercised no significant trans-regulation on the expression of the atp::lacZ fusions, indicating that the coupling is primarily cis. The mechanism of this coupling was investigated using in vitro mutagenesis. At least in the case of the pair atpHA, coupling seems to involve facilitated binding of fresh ribosomes to the atpA translational initiation regions.

Optimization of glucose oxidase production by Aspergillus niger using genetic- and process-engineering techniques.

Wild-type Aspergillus niger NRRL-3 was transformed with multiple copies of the glucose oxidase structural gene (god). The gene was placed under the control of the gpdA promoter of A. nidulans. For more efficient secretion the alpha-amylase signal peptide from A. oryzae was inserted in front of god. Compared to the wild type, the recombinant strain NRRL-3 (GOD3-18) produced up to four times more extracellular glucose oxidase under identical culture conditions. Addition of yeast extract (2 gl-1) to a mineral salts medium containing only glucose as carbon source increased volumetric and specific extracellular glucose oxidase activities by 130% and 50% respectively. With the same medium composition and inoculum size, volumetric and specific extracellular glucose oxidase activities increased more than ten times in bioreactor cultivations compared to shake-flask cultures.

Purification of protein A-tagged yeast ran reveals association with a novel karyopherin beta family member, Pdr6p.

The small GTPase Ran (encoded by GSP1 and GSP2 in yeast) plays a central role in nucleocytoplasmic transport. GSP1 and GSP2 were tagged with protein A and functionally expressed in a gsp1 null mutant. After affinity purification of protein A-tagged Gsp1p or Gsp2p by IgG-Sepharose chromatography, known karyopherin beta transport receptors (e.g. Kap121p and Kap123p) and a novel member of this protein family, Pdr6p, were found to be associated with yeast Ran. Subsequent tagging of Pdr6p with green fluorescent protein revealed association with the nuclear pore complexes in vivo. Thus, functional tagging of yeast Ran allowed the study of its in vivo distribution and interaction with known and novel Ran-binding proteins.

Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast.

The endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae contains of proteolytic system able to selectively degrade misfolded lumenal secretory proteins. For examination of the components involved in this degradation process, mutants were isolated. They could be divided into four complementation groups. The mutations led to stabilization of two different substrates for this process. The mutant classes were called 'der' for 'degradation in the ER'. DER1 was cloned by complementation of the der1-2 mutation. The DER1 gene codes for a novel, hydrophobic protein, that is localized to the ER. Deletion of DER1 abolished degradation of the substrate proteins. The function of the Der1 protein seems to be specifically required for the degradation process associated with the ER. The depletion of Der1 from cells causes neither detectable growth phenotypes nor a general accumulation of unfolded proteins in the ER. In DER1-deleted cells, a substrate protein for ER degradation is retained in the ER by the same mechanism which also retains lumenal ER residents. This suggests that DER1 acts in a process that directly removes protein from the folding environment of the ER.

The yeast protein Arc1p binds to tRNA and functions as a cofactor for the methionyl- and glutamyl-tRNA synthetases.

Arc1p was found in a screen for components that interact genetically with Los1p, a nuclear pore-associated yeast protein involved in tRNA biogenesis. Arc1p is associated with two proteins which were identified as methionyl-tRNA and glutamyl-tRNA synthetase (MetRS and GluRS) by a new mass spectrometry method. ARC1 gene disruption leads to slow growth and reduced MetRS activity, and synthetically lethal arc1- mutants are complemented by the genes for MetRS and GluRS. Recombinant Arc1p binds in vitro to purified monomeric yeast MetRS, but not to an N-terminal truncated form, and strongly increases its apparent affinity for tRNAMet. Furthermore, Arc1p, which is allelic to the quadruplex nucleic acid binding protein G4p1, exhibits specific binding to tRNA as determined by gel retardation and UV-cross-linking. Arc1p is, therefore, a yeast protein with dual specificity: it associates with tRNA and aminoacyl-tRNA synthetases. This functional interaction may be required for efficient aminoacylation in vivo.

Transcriptional profiling on all open reading frames of Saccharomyces cerevisiae.

Open reading frames (6116) of the budding yeast Saccharomyces cerevisiae were PCR-amplified from genomic DNA using 12,232 primers specific to the ends of the coding sequences; the success rate of amplification was 97%. PCR-products were made accessible to hybridization by being arrayed at very high density on solid support media using various robotic devices. Probes made from total RNA preparations were hybridized for the analysis of the transcriptional activity of yeast under various growth conditions and of different strains. Experimental factors that proved critical to the performance, such as different RNA isolation procedures and the assessment of hybridization results, for example, were investigated in detail. Various software tools were developed that permit convenient handling and sound analysis of the large data quantities obtained from transcriptional profiling studies. Comprehensive arrays are being distributed within the European Yeast Functional Analysis Network (EUROFAN) and beyond.

An expression system matures: a highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula polymorpha.

An efficient process was developed for the low-cost production of phytases using Hansenula polymorpha. Glucose or glucose syrups, previously reported as repressive substrates, were used as main carbon sources during fermentation. Glucose was even the most productive substrate for high-level production of phytases. Compared with the process using glycerol, the standard carbon source used for this process until now, the use of glucose led to a reduction of more than 80% in the raw materials costs. In addition, exceptionally high concentrations of active enzyme (up to 13.5 g/L) were obtained in the medium, with phytase representing over 97% of the total accumulated protein. These levels greatly exceed those reported so far for any yeast-based expression system. Very efficient downstream processing procedures were developed with product recovery yields over 90%. Both the fermentation and downstream processing were successfully tested in pilot scale up to 2000 L. As a result, H. polymorpha can be used as a highly competitive system for low-cost phytase production.