1H and 15N resonance assignments and secondary structure of the carbon monoxide complex of sperm whale myoglobin

Summary Sequence-specific backbone ¹H and ¹⁵N resonance assignments have been made for 95% of the amino acids in sperm whale myoglobin, complexed with carbon monoxide (MbCO). Many assignments for side-chain resonances have also been obtained. Assignments were made by analysis of an extensive series of homonuclear 2D spectra, measured with unlabeled protein, and both 2D and 3D ¹H-¹⁵N-correlated spectra obtained from uniformly ¹⁵N-labeled myoglobin. Patterns of medium-range NOE connectivities indicate the presence of eight helices in positions that are very similar to those found in the crystal structures of sperm whale myoglobin. The resonance assignments of MbCO form the basis for determination of the solution structure and for hydrogen-exchange measurements to probe the stability and folding pathways of myoglobin. They will also form a basis for assignment of the spectra of single-site mutants with altered ligand-binding properties.     

The evolution of pathways for aromatic hydrocarbon oxidation inPseudomonas

The organisation and nucleotide sequences coding for the catabolism of benzene, toluene (and xylenes), naphthalene and biphenylvia catechol and the extradiol (meta) cleavage pathway inPseudomonas are reviewed and the various factors which may have played a part in their evolution are considered. The data suggests that the complete pathways have evolved in a modular way probably from at least three elements. The commonmeta pathway operons, downstream from the ferredoxin-like protein adjacent to the gene for catechol 2,3-dioxygenase, are highly homologous and clearly share a common ancestry. This common module may have become fused to a gene or genes the product(s) of which could convert a stable chemical (benzoate, salicylate, toluene, benzene, phenol) to catechol, thus forming the lower pathway operons found in modern strains. The upper pathway operons might then have been acquired as a third module at a later stage thus increasing the catabolic versatility of the host strains.     

Solution structure by 1H and dynamics by natural abundance 13C NMR of a receptor recognising peptide derived from a C-terminal fragment of neuropeptide Y

Summary A peptide consisting of 20 amino acid residues, derived from a C-terminal fragment of neuropeptide Y (NPY) and showing high affinity to NPY receptors, was synthesised. Its sequence is PAADLARYRHYIN-LITRQRY-NH₂, and the solution structure was calculated from NMR-derived distance and torsion angle restraints, obtained at 15°C in a solvent mixture of water and 30% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol, by using DIANA and restrained energy minimisation. The structure was found to consist of a well-defined -helix in the centre, with a few residues at the termini having less well defined conformations. The spinlattice and spin-spin relaxation rates of -carbons have been determined on ¹³C at natural abundance. From 1D experiments the global rotational correlation time was determined and from 2D experiments the dynamics of each individual residue was obtained. The results demonstrate that the C-H vectors in the -helix essentially follow the global motion. Towards the termini, contributions from local dynamics increase. This tendency is correlated to the increasing uncertainty of the structure towards the peptide ends. An effective molecular volume was calculated from the temperature dependence of the global rotational correlation time. This is well compatible with a monomeric peptide, solvated by water and 1,1,1,3,3,3-hexafluoro-2-propanol. The presence of peptide dimers was ruled out as being inconsistent with the relaxation data.Supplementary material available from the authors: Two data tables and 10 PDB coordinate files of the calculated NMR structures of P7. One data table contains all detected and integrated NOE intensities; the other connects each proton and pseudoatom to an atom number used in the NOE table. The table contents served as input data files for CALIBA.Currently on leave from the Institute of Chemical Physics and Biophysics, Tallinn, Estonia.     

Localization of a cruciform cutting endonuclease to yeast mitochondria

We have found a cruciform cutting endonuclease in the yeast, Saccharomyces cerevisiae, which localizes to the mitochondria. This activity apparently is associated with the mitochondrial inner membrane since the activity is not released into solution by osmolysis, in contrast to the matrix enzyme, isocitrate dehydrogenase. The cruciform cutting activity appears to be encoded by CCE1. This gene has been shown to encode one of the major cruciform cutting endonucleases present in a yeast cell. In ccel strains, which lack CCE1 endonuclease activity, the mitochondrial cruciform cutting endonucleolytic activity is also absent. Since CCE1 is allelic to MGT1, a gene required for the highly biased transmission of petite mitochondrial DNA in crosses between ⁺ and hypersuppressive ^– cells, it seems likely that the CCE1 endonuclease functions within mitochondria.     

Enhanced detection of polymorphic DNA by multiple arbitrary amplicon profiling of endonuclease-digested DNA: identification of markers tightly linked to the supernodulation locus in soybean

Multiple endonuclease digestion of template DNA or amplification products can increase significantly the detection of polymorphic DNA in fingerprints generated by multiple arbitrary amplicon profiling (MAAP). This coupling of endonuclease cleavage and amplification of arbitrary stretches of DNA, directed by short oligonucleotide primers, readily allowed distinction of closely related fungal and bacterial isolates and plant cultivars. MAAP analysis of cleaved template DNA enabled the identification of molecular markers linked to a developmental locus of soybean (Glycine max L. Merrill). Ethyl methane sulfonate (EMS)-induced supernodulating, near-isogenic lines altered in the nts locus, which controls nodule formation, could be distinguished from each other and from the parent cultivar by amplification of template pre-digested with 2–3 restriction enzymes. A total of 42 DNA polymorphisms were detected using only 19 octamer primers. In the absence of digestion, 25 primers failed to differentiate these soybean genotypes. Several polymorphic products co-segregated tightly with the nts locus in F₂ families from crosses between the allelic mutants nts382 and nts1007 and the ancestral G. soja Sieb. & Succ. PI468.397. Our results suggest that EMS is capable of inducing extensive DNA alterations, probably around discrete mutational hot-spots. EMS-induced DNA polymorphisms may constitute sequence-tagged markers diagnostic of specific genomic regions.     

Population fragmentation may reduce fertility to zero in Banksia goodii — a demonstration of the Allee effect

All individuals of all known populations of Banksia goodii were assessed for seed production. Small populations produced no or only a few seeds per unit canopy area. Effects of population size on seed production per unit area and seed production per plant were present over the whole range of population sizes, indicating that even in large populations seed production may still not be at its maximum. Resource differences could not explain this disproportionate decrease in seed production with decline in population size, because there were no differences in soil properties and understorey or overstorey cover between the small and large populations. Although plants in small and large populations were similar in size, seed production per plant was much lower in small populations. This was not because plants in small populations produced fewer cones but because the fraction of these cones that was fertile was much lower. Five of the nine smallest populations (<200 m²) produced no fertile cones over the last 10 years. The number of seeds per fertile cone did not depend on population size. The results are discussed in relation to pollination biology.     

Size structure of the metazoan community in a Piedmont stream

We characterized the size structure of virtually the entire metazoan community in a fourth order, sandybottomed Piedmont stream during late summer. Our study, the first to sample across all habitat types and sizes of metazoans in an aquatic ecosystem, indicates that at the community level, stream size spectra may be bimodal for the benthos or trimodal when fish are included. Animals spanning 10 orders of magnitude in dry mass (from gastrotrichs to fish) were quantitatively collected from nine habitat types. The bimodal benthic size spectrum was characterized by a meiofaunal component (mostly oligochaetes and micro-crustacea) and a macrobenthic component (mostly the introduced asiatic clam, Corbicula fluminea). Insects contributed little to overall standing crop. Size-specific contribution to whole-community metabolism was assessed using allometric equations for respiration, and we found a distinctly bimodal distribution across the entire metazoan size range, with peaks in the meiofaunal and benthic macrofaunal size ranges. Our bimodal benthic size spectrum is similar to that observed for marine benthos but not to other freshwater benthic systems, possibly because the entire range of habitat types and/or animal sizes were not sampled in the latter. Numerous factors may influence size spectra in stream ecosystems, including local geomorphic (habitat) conditions, water level fluctuations, species introductions, and predation processes.     

Studies of 13C in the foliage reveal interactions between nutrients and water in forest fertilization experiments

The severity of a root rot disease of cereals, caused by Rhizoctonia solani Kühn AG8, was inversely correlated to the Zn status of plants in field studies in 1989 and 1990. In 1989, a preliminary survey was conducted in a farmer's field in South Australia where Zn deficiency and disease were both widespread. Zn concentration in Spear wheat plants at the 3-leaf to early tillering stage was negatively correlated with severity of the disease. For the elevent elements analysed, a correlation matrix showed that Zn had the highest, and only significant (R²=0.52^**) association with disease. The effect of Zn applications and their residual value on disease severity was further studied in a long-term field experiment in 1989 and 1990 to which Zn had been applied in 1986. There was a decrease in the area of Rhizoctonia bare patch as Zn rate was increased, a result consistent with the field survey results; the recommended rate of 2.5 kg Zn ha^–1 reduced the area affected by bare patch from 42% to 21% of the total crop area compared with no Zn application, overcame Zn deficiency and increased grain yield from 1.1 to 2.8 t ha^–1. In 1990, fresh Zn application treatments were applied to trial plots designed for this purpose, in order to compare the response with the older Zn treatments applied in 1986. The areas of bare patch in the older Zn treatments were approximately 5% greater than those in the fresh Zn treatments. The results are consistent with the hypothesis that Zn deficient plants are more susceptible to root rot caused by R. solani. Testing this hypothesis is the subject of a companion paper.     

Denitrification measurements in aquatic sediments: A comparison of three methods

Measurements of denitrification using the acetylene inhibition,¹⁵N isotope tracer, and N₂ flux methods were carried out concurrently using sediment cores from Vilhelmsborg sø, Denmark, in an attempt to clarify some of the limitations of each technique. Three experimental treatments of overlying water were used: control, nitrate enriched, and ammonia enriched water. The N₂ flux and¹⁵N tracer experiments showed high rates of coupled nitrification/denitrification in the sediments. The acetylene inhibition method did not capture any coupled nitrification/denitrification. This could be explained by acetylene inhibition of nitrification. A combined¹⁵N tracer/acetylene inhibition experiment demonstrated that acetylene inhibition of N₂O reduction was incomplete and the method, therefore, only measured approximately 50% of the denitrification due to nitrate from the overlying water. Similar rates of denitrification due to nitrate in the overlying water were measured by the N₂ flux method and the acetylene inhibition method, after correcting for the 50% efficiency of acetylene inhibition. Rates of denitrification due to nitrate from the overlying water measured by the¹⁵N tracer method, however, were only approximately 35% or less of those measured by the acetylene inhibition or N₂ flux methods.