Dominance and recessiveness at the protein level in mutant x wildtype crosses in Sacchaomyces cerevisiae

Summary The is ₁-locus of the yeast Saccharomyces cerevisiae is the structural gene for threonine dehydratase. is ₁-mutants require isoleucine for growth and do not have active threonine dehydratase.Interallelic complementation is frequent among is ₁-mutants. This is indicative for an aggregate or multimeric structure of yeast threonine dehydratase.Complementing and non-complementing mutants were crossed to wildtype. Properties of threonine dehydratase were assayed in crude extracts of the resulting heterozygotes.Specific activities varied considerably between full wildtype activity and a level about 10% of that. The apparent Michaelis constants were increased in many heterozygotes. This effect was probably due to the aggregation of both mutant and wildtype subunits to form a hybrid threonine dehydratase with reduced substrate affinity in addition to pure wildtype enzyme. This notion is supported by the observation in one heterozygote of two enzyme fractions with increased Michaelis constants in addition to a wildtype-like fraction.The possible formation of hybrid enzymes with normal, reduced or no activity is considered to blur gene dosage relations.A given pair of alleles in a heterozygous cell can generate a new type of enzyme with properties not encountered in the corresponding two homozygous cells. This situation is not accounted for by the classical concepts of dominant-recessive or intermediate behaviour, because the difference between the heterozygotes and the homozygotes is not necessarily only quantitativ but also qualitative.We dedicate this publication to Prof. Dr. C. Auerbach on occasion of her official retirement in admiration for her pioneer work and many contribution to genetics.     

The HOPS Proteins hVps41 and hVps39 Are Required for Homotypic and Heterotypic Late Endosome Fusion

The homotypic fusion and protein sorting (HOPS) complex is a multisubunit tethering complex that in yeast regulates membrane fusion events with the vacuole, the yeast lysosome. Mammalian homologs of all HOPS components have been found, but little is known about their function. Here, we studied the role of hVps41 and hVps39, two components of the putative human HOPS complex, in the endo‐lysosomal pathway of human cells. By expressing hemagglutinin (HA)‐tagged constructs, we show by immunoelectron microscopy (immunoEM) that both hVps41 and hVps39 associate with the limiting membrane of late endosomes as well as lysosomes. Small interference RNA (siRNA)‐mediated knockdown of hVps41 or hVps39 resulted in an accumulation of late endosomes, a depletion in the number of lysosomes and a block in the degradation of endocytosed cargo. Lysosomal pH and cathepsin B activity remained unaltered in these conditions. By immunoEM we found that hVps41 or hVps39 knockdown impairs homotypic fusion between late endosomes as well as heterotypic fusion between late endosomes and lysosomes. Thus, our data show that both hVps41 and hVps39 are required for late endosomal–lysosomal fusion events and the delivery of endocytic cargo to lysosomes in human cells.     

High Sensitive Detection of Carbohydrate Binding Proteins in an ELISA-Solid Phase Assay Based on Multivalent Glyconanoparticles

Improved detection of anti-carbohydrate antibodies is a need in clinical identification of biomarkers for cancer cells or pathogens. Here, we report a new ELISA approach for the detection of specific immunoglobulins (IgGs) against carbohydrates. Two nanometer gold glyconanoparticles bearing oligosaccharide epitopes of HIV or Streptococcus pneumoniae were used as antigens to coat ELISA-plates. A ~3,000-fold improved detection of specific IgGs in mice immunized against S. pneumoniae respect to the well known BSA-glycoconjugate ELISA was achieved. Moreover, these multivalent glyconanoparticles have been employed in solid phase assays to detect the carbohydrate-dependent binding of human dendritic cells and the lectin DC-SIGN. Multivalent glyconanoparticles in ELISA provide a versatile, easy and highly sensitive method to detect and quantify the binding of glycan to proteins and to facilitate the identification of biomarkers.     

Herbivore trampling as an alternative pathway for explaining differences in nitrogen mineralization in moist grasslands

Studies addressing the role of large herbivores on nitrogen cycling in grasslands have suggested that the direction of effects depends on soil fertility. Via selection for high quality plant species and input of dung and urine, large herbivores have been shown to speed up nitrogen cycling in fertile grassland soils while slowing down nitrogen cycling in unfertile soils. However, recent studies show that large herbivores can reduce nitrogen mineralization in some temperate fertile soils, but not in others. To explain this, we hypothesize that large herbivores can reduce nitrogen mineralization in loamy or clay soils through soil compaction, but not in sandy soils. Especially under wet conditions, strong compaction in clay soils can lead to periods of soil anoxia, which reduces decomposition of soil organic matter and, hence, N mineralization. In this study, we use a long-term (37-year) field experiment on a salt marsh to investigate the hypothesis that the effect of large herbivores on nitrogen mineralization depends on soil texture. Our results confirm that the presence of large herbivores decreased nitrogen mineralization rate in a clay soil, but not in a sandy soil. By comparing a hand-mown treatment with a herbivore-grazed treatment, we show that these differences can be attributed to herbivore-induced changes in soil physical properties rather than to above-ground biomass removal. On clay soil, we find that large herbivores increase the soil water-filled porosity, induce more negative soil redox potentials, reduce soil macrofauna abundance, and reduce decomposition activity. On sandy soil, we observe no changes in these variables in response to grazing. We conclude that effects of large herbivores on nitrogen mineralization cannot be understood without taking soil texture, soil moisture, and feedbacks through soil macrofauna into account.     

Feeding behaviour and performance of different populations of the black currant‐lettuce aphid,Nasonovia ribisnigri,on resistant and susceptible lettuce

When crops are bred for resistance to herbivores, these herbivores are under strong selection pressure to overcome this resistance, which may result in the emergence of virulent biotypes. This is a growing problem for crop species attacked by aphids. The Nr‐gene in lettuce confers near‐complete resistance against the black currant‐lettuce aphid, Nasonovia ribisnigri (Mosely) (Hemiptera: Aphididae). Since 2007, populations of N. ribisnigri have been reported in several locations in Europe to infest resistant lettuce varieties that possess the Nr‐gene. The objective of this study was to analyse the behaviour and level of virulence of several N. ribisnigri populations observed to have colonized Nr‐locus‐containing lettuce lines. We analysed the stylet penetration and feeding behaviour, and the performance of these N. ribisnigri populations on resistant and susceptible lettuce lines. Large variation in the degree of virulence to the Nr‐locus‐containing lettuce lines was found among populations of the Nr:1 biotype. The German population was highly virulent on the Nr‐containing resistant lettuce lines, and showed similar feeding behaviour and performance on both the susceptible and resistant lettuces. The French population from Paris was the second most virulent, though reproduction on the resistant lines was reduced. The French population from Perpignan and a population from Belgium, however, showed reduced performance and feeding rate on the resistant compared to the susceptible lettuces. The lettuce background in which the Nr‐gene is expressed influences the level of resistance to the various Nr:1 aphid populations, because the performance and feeding behaviour differed between the aphids on the cultivars (romaine lettuce) compared to the near‐isogenic lines (butterhead/iceberg lettuce). This study also shows that being able to feed on a plant not automatically implies that a population can successfully develop on that plant, because aphids showed phloem ingestion during the 8‐h recording period on resistant lettuce, but were not able to survive and reproduce on the same lettuce line.