Generation of New Hairless Alleles by Genomic Engineering at the Hairless Locus in Drosophila melanogaster

Hairless (H) is the major antagonist within the Notch signalling pathway of Drosophila melanogaster. By binding to Suppressor of Hairless [Su(H)] and two co-repressors, H induces silencing of Notch target genes in the absence of Notch signals. We have applied genomic engineering to create several new H alleles. To this end the endogenous H locus was replaced with an attP site by homologous recombination, serving as a landing platform for subsequent site directed integration of different H constructs. This way we generated a complete H knock out allele H ^attP, reintroduced a wild type H genomic and a cDNA-construct (H ^gwt, H ^cwt) as well as two constructs encoding H proteins defective of Su(H) binding (H ^LD, H ^iD). Phenotypes regarding viability, bristle and wing development were recorded, and the expression of Notch target genes wingless and cut was analysed in mutant wing discs or in mutant cell clones. Moreover, genetic interactions with Notch (N ⁵⁴¹⁹) and Delta (Dl ^B2) mutants were addressed. Overall, phenotypes were largely as expected: both H ^LD and H ^iD were similar to the H ^attP null allele, indicating that most of H activity requires the binding of Su(H). Both rescue constructs H ^gwt and H ^cwt were homozygous viable without phenotype. Unexpectedly, the hemizygous condition uncovered that they were not identical to the wild type allele: notably H ^cwt showed a markedly reduced activity, suggesting the presence of as yet unidentified regulatory or stabilizing elements in untranslated regions of the H gene. Interestingly, H ^gwt homozygous cells expressed higher levels of H protein, perhaps unravelling gene-by-environment interactions.     

Role of Asp297 of the AT2 receptor in high-affinity binding to different peptide ligands

To determine how ligand-receptor interaction is affected by the charges of the amino acids at position 2 of the ligands and position 297 of the AT2 receptor, we generated the Asp297Lys mutant of AT2 and a ligand SarAsp²Ile. Asp297Lys mutant lost affinity to Ang II and SarIle however retained partial affinity to ¹²⁵I-CGP42112A. The SarAsp²Ile had high affinity to Asp297Lys (IC₅₀3.5nM) and partial affinity to the AT2 (IC₅₀15nM). Therefore, not only the charge, but also the length of the side arms of the amino acids at position 2 of the ligand and position 297 of the receptor affect their interaction.     

Modellversuche zur überlebensdauer von Erwinia amylovora im Verdauungstrakt der Honigbiene,im Honig und an Teilen des Bienenstocks

An lagerndem Winterknoblauch ist unter außenluft‐(x + 6, 5 bzw. 8, 3 °C) und maschinengekühlten Bedingungen (x‐1 . . .‐2 °C) in zwei Lagerperioden die Entwicklung der einzelnen Fäuleerreger verfolgt und in Abhängigkeit von der Lagerdauer statistisch quantifiziert worden. An Hand des Masseanteiles befallener Zwiebeln und der Befallsintensität wird eine zunehmende Ausbreitung von Penicillium spp. und einer Gruppe mit Befall durch mehrere Fäuleerreger unter beiden Lagerbedingungen und von einer Botrytis‐Species (vermutlich B. porri Buchw.) im maschinengekühlten Lager belegt. Fäuleverluste durch Helminthosporium allii Campanile und Bakterien zeigen dagegen mit fortschreitender Lagerdauer einen abnehmenden Verlauf. Mit Eintreten von lagerungsbedingter Seneszenz steigen die Verluste progressiv an. Kaltlagerbedingungen verzögern dagegen ihre Ausbreitung. Die Verluste durch alle Fäulerreger (Fäule gesamt) zeigen eine gleichmäßige Zunahme während der Lagerdauer.     

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cell. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ‘Crabtree effect’, was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate hydrogenase was subject to glucose inactivation.     

Schiffs bases and derived secondary amines as plant growth inhibitors

Seventy-two Schiffs bases, 44 corresponding secondary amines, and 12 N-acetylated compounds were tested on their growth activity. Eighty-one compounds were active as growth inhibitors in at least one of three bioassays.     

Flavonoid and anthocyanin patterns and the systematic relationships in Collomia

Analyses of extracts among populations of the 14 species of Collomia revealed the occurrence of 13 mono-, di- and triglycosides based on the flavonoids, acacetin, kaempferol, patuletin and quercetin. The glycosides included those having arabinose, galactose, glucose and rhamnose as mono-, bio- or triosides at the 3-, 5-, 3,7- or 7-position. Analyses of floral extracts from ten species revealed the occurrence of two anthocyanins, cyanidin and delphinidin 3-(p-coumarylglucosyl)-5-glucoside. Nearly all the species express distinctive flavonoid patterns, although the differences are based on relatively minor changes in position or type of glycosidic substitution. Use of the minimum biosynthetic step distance (MBSD), an index of similarity, revealed that a mean of 5.6 steps separated the 14 species. The four perennial species of section Collomiastrum showed a high degree of similarity and differed consistently from species of the two annual sections Courtoisia and Collomia by lacking quercetin-5-glucoside and kaempferol-3-arabinosylgalactoside. In contrast, flavonoid patterns among species within sections Courtoisia and Collomia showed a relatively low degree of similarity. The dissimilarity between C. diversifolia and C. heterophylla (section Courtoisia) is consistent with their divergent patterns of pollen morphology and ecological distribution. Three groups of species within section Collomia were defined generally by shared patterns of flavonoids, which are correlated to some degree with floral, pollen and vegetative morphology.     

Mutational analysis of the carbohydrate binding activity of the tobacco lectin

At present the three-dimensional structure of the tobacco lectin, further referred to as Nictaba, and its carbohydrate-binding site are unresolved. In this paper, we propose a three-dimensional model for the Nictaba domain based on the homology between Nictaba and the carbohydrate-binding module 22 of Clostridium thermocellum Xyn10B. The suggested model nicely fits with results from circular dichroism experiments, indicating that Nictaba consists mainly of β-sheet. In addition, the previously identified nuclear localization signal is located at the top of the protein as a part of a protruding loop. Judging from this model and sequence alignments with closely related proteins, conserved glutamic acid and tryptophan residues in the Nictaba sequence were selected for mutational analysis. The mutant DNA sequences as well as the original Nictaba sequence have been expressed in Pichia pastoris and the recombinant proteins were purified from the culture medium. Subsequently, the recombinant proteins were characterized and their carbohydrate binding properties analyzed with glycan array technology. It was shown that mutation of glutamic acid residues in the C-terminal half of the protein did not alter the carbohydrate-binding activity of the lectin. In contrast, mutation of tryptophan residues in the N-terminal half of the Nictaba domain resulted in a complete loss of carbohydrate binding activity. These results suggest that tryptophan residues play an important role in the carbohydrate binding site of Nictaba.     

Presence and Analysis of Plasmids in Human and Animal Associated Arcobacter Species

In this study, we report the screening of four Arcobacter species for the presence of small and large plasmids. Plasmids were present in 9.9% of the 273 examined strains. One Arcobacter cryaerophilus and four Arcobacter butzleri plasmids were selected for further sequencing. The size of three small plasmids isolated from A. butzleri and the one from A. cryaerophilus strains ranged between 4.8 and 5.1 kb, and the size of the large plasmid, isolated from A. butzleri, was 27.4 kbp. The G+C content of all plasmids ranged between 25.4% and 26.2%. A total of 95% of the large plasmid sequence represents coding information, which contrasts to the 20 to 30% for the small plasmids. Some of the open reading frames showed a high homology to putative conserved domains found in other related organisms, such as replication, mobilization and genes involved in type IV secretion system. The large plasmid carried 35 coding sequences, including seven genes in a contiguous region of 11.6 kbp that encodes an orthologous type IV secretion system found in the Wolinella succinogenes genome, Helicobacter pylori and Campylobacter jejuni plasmids, which makes this plasmid interesting for further exploration.