Ten polymorphic microsatellite loci identified from a small insert genomic library for Peronospora tabacina

Ten polymorphic microsatellite loci for the obligate biotrophic, oomycete pathogen of tobacco, Peronospora tabacina, were identified from a small insert genomic library enriched for GT motifs. Eighty-five percent of the 162 loci identified were composed of dinucleotide repeats, whereas only 4% and 11% were tri-and tetra-nucleotide repeats respectively. About 82% of all the microsatellites were perfect and within the library; only about 7% of the loci were duplicated. Primers were designed for 63 loci; 10 loci were polymorphic, 19 were monomorphic and 34 either failed to amplify or produced ambiguous/inconsistent results. The 10 polymorphic loci were characterized with 44 isolates of P. tabacina collected from tobacco plants growing in Europe, the Near East and North and South America. The number of alleles per locus was either three or four with a mean of 3.2, and the mean number of genotypes per locus was 3.6. Observed heterozygosity was 0.32-0.95, whereas expected heterozygosity was 0.44-0.69 for these loci. All loci except PT054 did not conform to the Hardy-Weinberg distribution. Polymorphic information content (PIC) for the loci was 0.35-0.69 with a mean of 0.50. These microsatellite loci provide a set of markers sufficient to perform genetic diversity and population studies of P. tabacina, and possibly other species of Peronospora.     

The buffering capacity of the internal phase of thylakoids and the magnitude of the pH changes inside under flashing light.

The buffering capacity inside thylakoids is determined and the magnitude of flash-induced pH changes inside is calibrated in the pH range from 6.4 to 8.1. The work is based on flash-induced absorption changes of neutral red in a chloroplast suspension in which the outer phase is strongly buffered by bovine serum albumin. It is shown that neutral red is bound inside thylakoids. The binding can be described by a simple isotherm with an apparent Km = 4 microM and satruation at 1 neutral red per 17 chlorophylls. The apparent pK of neutral red is shifted from 6.6 in solution to 7.25 when bound inside. It is demonstrated that neutral red is a clean indicator of pH changes inside, i.e. when properly used it shows no response to other events. Although bound it reports pH changes which occur in the internal osmolar (aqueous) volume of thylakoids. This is obvious from the influence of chemically very different buffers on the magnitude of the absorption changes of neutral red. These act in a manner proportional to their calculated buffering capacity in aqueous solution. The intrinsic buffering capacity of the internal phase is determined with the aid of these buffers, at pH 7.2 it is between 0.8 and 1 mM (at 60 mosM). The absence of large variations in the buffering capacity in the range from pH 6.4 to 8.1 suggests that proteinaceous groups are involved in addition to the lipids which may dominate the buffering capacity at lower pH. The magnitude of the internal pH change is arrpox. 0.6 (at pH 7.3) under stimulation of both photosystems with a short xenon flash of light.     

A new yeast mutation in the glucosylation steps of the asparagine-linked glycosylation pathway. Formation of a novel asparagine-linked oligosaccharide containing two glucose residues

We have isolated and characterized a new yeast mutation in the glucosylation steps of lipid-linked oligosaccharide biosynthesis, alg8-1. Cells carrying the alg8-1 mutation accumulate Glc1Man9GlcNAc2-lipid both in vivo and in vitro. We present evidence showing that the alg8-1 mutation blocks addition of the second alpha 1,3-linked glucose. alg8-1 cells transfer Glc1Man9GlcNAc2 to protein instead of the wild type oligosaccharide, Glc3Man9GlcNAc2. Pulse-chase studies indicate that the Glc1Man9GlcNAc2 transferred is processed more slowly than the wild type oligosaccharide. The yeast mutation gls1-1 lacks glucosidase I activity (Esmon, B., Esmon, P.C., and Schekman, R. (1984) J. Biol. Chem. 259, 10322-10327), the enzyme responsible for removing the alpha 1,2-linked glucose residues from protein-linked oligosaccharides. We demonstrate that gls1-1 cells contain glucosidase II activity (which removes alpha 1,3-linked glucose residues) and have constructed the alg8-1 gls1-1 haploid double mutant. The Glc1Man9GlcNAc2 oligosaccharide was trimmed normally in these cells, demonstrating that the alg8-1 oligosaccharide contained an alpha 1,3-linked glucose residue. A novel Glc2 compound was probably produced by the action of the biosynthetic enzyme that normally adds the alpha 1,2-linked glucose to lipid-linked Glc2Man9GlcNAc2. This enzyme may be able to slowly add alpha 1,2-linked glucose residue to protein-bound Glc1Man9GlcNAc2. The relevance of these findings to similar observations in other systems where glucose residues are added to asparagine-linked oligosaccharides and the possible significance of the reduced rate of oligosaccharide trimming in the alg mutants are discussed.     

Jährliche Schwankungen Der Individuenzahl In Einer Nordwestdeutschen Trockenen Heide III

Summary The vegetation of a permanent quadrat situated in a Calluno-Genistetum typicum community has been analysed every year from 1955–1964 (cf. Vegetatio Vol. X, Fasc. 1, 1961 and Vol. XIII, Fasc. 4, 1966) and again from 1965 to 1969. Calluna vulgaris, which from 1955 to 1959 still covered 85–95 % of the area, decreased to 30–55 %. This was due to disease or perhaps was a result of sheep grazing. On the other hand,Betula pendula, which had appeared first in 1961, has increased continually in the permanent quadrat. This increase is probably due to the sheep, who avoid the young trees ofBetula pendula. The number and cover of the other plants fluctuated due to variations in climate or the sheep grazing.      

Protein-tyrosine phosphatase (PTP) wedge domain peptides: a novel approach for inhibition of PTP function and augmentation of protein-tyrosine kinase function

Inhibition of protein-tyrosine phosphatases (PTPs) counterbalancing protein-tyrosine kinases (PTKs) offers a strategy for augmenting PTK actions. Conservation of PTP catalytic sites limits development of specific PTP inhibitors. A number of receptor PTPs, including the leukocyte common antigen-related (LAR) receptor and PTPmu, contain a wedge-shaped helix-loop-helix located near the first catalytic domain. Helix-loop-helix domains in other proteins demonstrate homophilic binding and inhibit function; therefore, we tested the hypothesis that LAR wedge domain peptides would exhibit homophilic binding, bind to LAR, and inhibit LAR function. Fluorescent beads coated with LAR or PTPmu wedge peptides demonstrated PTP-specific homophilic binding, and LAR wedge peptide-coated beads precipitated LAR protein. Administration of LAR wedge Tat peptide to PC12 cells resulted in increased proliferation, decreased cell death, increased neurite outgrowth, and augmented Trk PTK-mediated responses to nerve growth factor (NGF), a phenotype matching that found in PC12 cells with reduced LAR levels. PTPmu wedge Tat peptide had no effect on PC12 cells but blocked the PTPmu-dependent phenotype of neurite outgrowth of retinal ganglion neurons on a PTPmu substrate, whereas LAR wedge peptide had no effect. The survival- and neurite-promoting effect of the LAR wedge peptide was blocked by the Trk inhibitor K252a, and reciprocal co-immunoprecipitation demonstrated LAR/TrkA association. The addition of LAR wedge peptide inhibited LAR co-immunoprecipitation with TrkA, augmented NGF-induced activation of TrkA, ERK, and AKT, and in the absence of exogenous NGF, induced activation of TrkA, ERK, and AKT. PTP wedge domain peptides provide a unique PTP inhibition strategy and offer a novel approach for augmenting PTK function.