Heavy riboflavin synthase from Bacillus subtilis. Particle dimensions, crystal packing and molecular symmetry

Heavy riboflavin synthase from Bacillus subtilis is an enzyme complex consisting of approximately three alpha-subunits (Mr 23.5 X 10(3)) and 60 beta-subunits (Mr 16 X 10(3)). The enzyme has been crystallized from phosphate buffer in a hexagonal crystal modification that belongs to space group P6(3)22. The asymmetric unit of the crystal cell contains ten beta-subunits. The structure of this unusual 10(6) Mr protein has been studied by small-angle X-ray scattering, electron microscopy of three-dimensional crystals, and crystallographic methods. The scattering curves can be interpreted in terms of a hollow sphere model with a ratio of inner and outer radius of 0.3:1. A diameter of 168 A was estimated from the scattering curves, in close agreement with electron microscopic studies. An aggregate with the stoichiometry beta 60, which was obtained by ligand-driven reaggregation of isolated beta-subunits, showed similar shape and dimensions, but a larger value for the ratio Ri/Ra. Electron micrographs of freeze-etched enzyme crystals showed approximately spherical molecules, which were arranged in hexagonal layers. The lattice constants found from the micrographs are in good agreement with the values derived from X-ray diffraction data. Rotation function calculations in Patterson space showed a set of peaks for 2-fold, 3-fold and 5-fold local rotation axes, accurately consistent with icosahedral symmetry and with the particle orientation A shown in the Appendix. The crystal packing can be described as follows: enzyme particles with icosahedral symmetry (point group 532) are located at points 32 of the hexagonal cell, corresponding to positions (0, 0, 0) and (0, 0, 1/2) on the 6-fold screw axes. From the data reported, it may be concluded that the enzyme structure can be described as an icosahedral capsid of 60 beta-subunits with the triangulation number T = 1. The alpha-subunits are located in the central core space of the capsid, but their spatial orientation is incompletely understood.     

Mature mRNA is selectively released from the nuclear matrix by an ATP/dATP-dependent mechanism sensitive to topoisomerase inhibitors

Ovalbumin mRNA precursors were found to be almost quantitatively associated with the hen oviduct nuclear matrix. On the other hand, only one-third of the mature ovalbumin mRNA of whole nuclei was recovered in the nuclear matrix fraction. The binding of both the high molecular weight mRNA precursors and the mature-sized mRNA to the matrix displayed no difference in stability against salt, urea, or detergents. The mature mRNA, however, was found to be released selectively from the matrix by ATP. In contrast, the mRNA precursors remained completely bound to the nuclear substructure in the presence of ATP. Detachment of mRNA from the matrix also occurred in the presence of ADP, AMP plus pyrophosphate, or ATP analogs that contain nonhydrolyzable alpha, beta and beta, gamma bonds. Contrasting with the ATP-induced effect, addition of poly(A), ethidium bromide, or the copper chelator 1,10-phenanthroline to oviduct cell matrices caused an unspecific liberation of both mature and immature ovalbumin messengers. The release of the mature mRNA by ATP was found to be strongly inhibited by both nonintercalative and intercalative inhibitors of type II topoisomerase. These results suggest that the selection of the mature mRNAs for nucleocytoplasmic transport occurs at the release stage from the matrix (i.e. before translocation through the nuclear pore) and that reactions hitherto known to cause changes in the DNA secondary structure are associated with the detachment of mRNA from the nuclear substructure.     

Eine neue Methode zur Messung des relativen Lichtgenusses der Pflanzen

Ohne ZusammenfassungMit 7 Textabbildungen.     

Aerobic biomineralization of alpha-hexachlorocyclohexane in contaminated soil

The factors identified to be important for the aerobic biodegradation of alpha-hexachlorocyclohexane (alpha-HCH) in a soil slurry are temperature, auxiliary carbon source, substrate concentration, and soil inhomogeneities. Temperatures in the range of 20 to 30 degrees C were determined to be most favorable for biodegradation of alpha-HCH. No alpha-HCH biodegradation was detected at temperatures below 4 degrees C and above 40 degrees C. The addition of auxiliary organic carbon compounds showed repressive effects on alpha-HCH biomineralization. Increased oxygen partial pressures reduced the repressive effects of added auxiliary organic carbon compounds. A linear relationship between alpha-HCH concentration and its conversion rate was found in a Lineweaver-Burk plot. Inhomogeneities such as clumping of alpha-HCH significantly affected its biodegradation. Inhomogeneity as an influence on biodegradation has not drawn sufficient attention in the past, even though it certainly has affected both laboratory studies and the application of biotechnological methods to clean up contaminated sites. On the basis of metabolites detected during degradation experiments, the initial steps of aerobic alpha-HCH bioconversion in a soil slurry are proposed.     

Influence of amino acids,mammalian serum,and osmotic pressure on the proliferation of Drosophila cell lines

In the D22 medium of ECHALIER and OHANESSIAN for the culture of Drosophila cell lines lactalbumin hydrolysate could be replaced by a synthetic amino acids mixture. In spite of the presence of yeast extract and fetal calf serum the omission of any one of arginine, asparagine, cysteine, histidine, methionine, proline, serine, or threonine prevented cell proliferation. Of these eight amino acids cysteine had to be added in concentrations higher than 0.1 mM. Without much effect on cell proliferation foetal calf serum could be reduced from 10% to 2% or be replaced by 1% horse serum or 1% porcine serum. Cells could grow in media of osmolarities from 225 mOsm up to 400 mOsm depending on the osmotic agent used. Chloride concentrations up to 80 mM were compatible with proliferation as was a wide range of sodium/potassium ratios.     

A Non-Synonymous HMGA2 Variant Decreases Height in Shetland Ponies and Other Small Horses

The identification of quantitative trait loci (QTL) such as height and their underlying causative variants is still challenging and often requires large sample sizes. In humans hundreds of loci with small effects control the heritable portion of height variability. In domestic animals, typically only a few loci with comparatively large effects explain a major fraction of the heritability. We investigated height at withers in Shetland ponies and mapped a QTL to ECA 6 by genome-wide association (GWAS) using a small cohort of only 48 animals and the Illumina equine SNP70 BeadChip. Fine-mapping revealed a shared haplotype block of 793 kb in small Shetland ponies. The HMGA2 gene, known to be associated with height in horses and many other species, was located in the associated haplotype. After closing a gap in the equine reference genome we identified a non-synonymous variant in the first exon of HMGA2 in small Shetland ponies. The variant was predicted to affect the functionally important first AT-hook DNA binding domain of the HMGA2 protein (c.83G>A; p.G28E). We assessed the functional impact and found impaired DNA binding of a peptide with the mutant sequence in an electrophoretic mobility shift assay. This suggests that the HMGA2 variant also affects DNA binding in vivo and thus leads to reduced growth and a smaller stature in Shetland ponies. The identified HMGA2 variant also segregates in several other pony breeds but was not found in regular-sized horse breeds. We therefore conclude that we identified a quantitative trait nucleotide for height in horses.     

Julius Sachs

Ohne Zusammenfassung