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J Piefke

Publications and source records attributed to J Piefke.

12 recordsLinked to original sources
PubMed2001

Advanced integrated mouse YAC map including BAC framework.

Functional characterization of the mouse genome requires the availability of a comprehensive physical map to obtain molecular access to chromosomal regions of interest. Positional cloning remains a crucial way of linking phenotype with particular genes. A key step and frequent stumbling block in positional cloning is making a contig of a genetically defined candidate region. The most efficient first step is isolating YAC (Yeast Artificial Chromosome) clones. A robust, detailed YAC contig map is thus an important tool. Employing Interspersed Repetitive Sequence (IRS)-PCR genomics, we have generated an advanced second-generation YAC contig map of the mouse genome that doubles both the depth of clones and the density of markers available. In addition to the primarily YAC-based map, we located 1942 BAC (Bacterial Artificial Chromosome) clones. This allows us to present for the first time a dense framework of BACs spanning the genome of the mouse, which, for instance, can serve as a nucleus for genomic sequencing. Four large-insert mouse YAC libraries from three different strains are included in our data, and our analysis incorporates the data of Hunter et al. and Nusbaum et al. There is a total of 20,205 markers on the final map, 12,033 from our own data, and a total of 56,093 YACs, of which 44,401 are positive for more than one marker.

Algorithms↗
PubMed1994

Electron cryomicroscopy of Bacillus stearothermophilus 50 S ribosomal subunits crystallized on phospholipid monolayers.

50 S ribosomal subunits from Bacillus stearothermophilus have been crystallized as 2-dimensional periodic arrays on phospholipid monolayer films at the water-air interface. These crystals were preserved in vitreous ice and imaged with 100 keV electrons under low dose and low temperature conditions. The unit cell parameters of the crystals are a = 371.3(+/- 3.8) A, b = 152.3(+/- 1.6) A, gamma = 96.3(+/- 1.0) degrees. Some of the image arrays of these crystals have twofold rotational symmetry with a phase residual of less than 25 degrees. The mean figure of merit of the merged structure factors from these image arrays out to 20 A resolution is higher than 0.87. The 2-dimensional projection map shows a level of detail not seen in previous structural studies of the 50 S ribosome subunit. Some of these features may be related to the current 3-dimensional model of the subunit. This analysis illustrates the potential of using the electron crystallographic approach for determining the 3-dimensional structure of the 50 S ribosomal subunit crystallized on a monolayer surface. In addition, the structural information retrieved by electron crystallography might be useful for phasing X-ray data towards an atomic resolution model of the ribosome.

Crystallography↗
PubMed1990

Crystals of complexes mimicking protein biosynthesis are suitable for crystallographic studies.

A complex of 70S ribosomes from Thermus thermophilus together with an average of 1.5-1.8 equivalents of PhetRNA(Phe) and a short mRNA chain, composed of 35 +/- 5 uridines, was crystallized under the conditions used for the growth of crystals of isolated ribosomes from the same source. Considering the reproducibility of their growth, their internal order and their shape, the crystals of the complex are superior to those of isolated ribosomes. In accord with previous three-dimensional reconstruction and modeling experiments, we conclude that the complex is less flexible and that an average population of complexes is more homogeneous than that of isolated 70S ribosomes. The crystals of the complex diffract to higher than 15 A resolution and can be irradiated with synchrotron X-ray beam at cryo-temperatures for days without noticeable decay. Since the crystals of the complex are apparently isomorphous with these of the isolated 70S ribosomes (P4(1)2(1)2; a = b = 526; c = 315 A), they should provide tool for phasing as well as for locating the mRNA and tRNA binding sites.

Computer Graphics↗
PubMed1988

Two-dimensional crystalline sheets of Bacillus stearothermophilus 50S ribosomal subunits containing a nascent polypeptide chain.

Polylysine chains were synthesized on Bacillus stearothermophilus ribosomes in a poly(A)-programmed in vitro system. After separation of the ribosomal subunits by sucrose gradient centrifugation, the polylysine chains (in contrast to the polyphenylalanine chains synthesized in a poly(U) system) reproducibly remained attached to the large ribosomal subunit. It was possible to produce two-dimensional crystalline sheets from the large ribosomal subunits containing the polylysine chains. These sheets are an essential prerequisite for three-dimensional reconstruction studies aiming to show that the tunnel in the large ribosomal subunit provides a path for the nascent polypeptide chain.

Cell Fractionation↗
PubMed1987

The growth of ordered two-dimensional sheets of ribosomal particles from salt-alcohol mixtures.

A procedure for the in vitro growth of well-ordered two-dimensional sheets from ribosomal particles using salts and salt-alcohol mixtures has been developed. Employing this procedure, ordered two-dimensional sheets of the wild type as well as of mutated 50 S ribosomal subunits from Bacillus stearothermophilus can readily be obtained. These sheets, stained with uranyl acetate or gold-thioglucose, are suitable for three-dimensional image reconstruction. They consist of relatively small unit cells with dimensions of 160 +/- 15 and 365 +/- 20 A. Diffraction patterns of electron micrographs of these sheets contain features to 25 A resolution.

Crystallization↗
PubMed1987

Three-dimensional image reconstruction from ordered arrays of 70S ribosomes.

A better understanding of the molecular mechanism of protein biosynthesis still awaits a reliable model for the ribosomal particle. We describe here the application of a diffraction technique, namely three-dimensional image reconstruction from two-dimensional sheets of 70S ribosomes from Bacillus stearothermophilus at 47 A resolution. The three-dimensional model obtained by these studies shows clearly the two subunits, the contact points between them, an empty space large enough to accommodate the components of protein biosynthesis, the location of regions rich in RNA and a possible binding site for mRNA. The tunnel within the 50S particle which may provide the path taken by the nascent polypeptide chain in partially resolved.

Geobacillus stearothermophilus↗
PubMed1986

The growth of ordered two-dimensional sheets of 70 S ribosomes from Bacillus stearothermophilus.

Well ordered two-dimensional sheets of intact 70 S ribosomes from Bacillus stearothermophilus have been obtained in vitro using salt-alcohol mixtures. These sheets consist of relatively small unit cells with dimensions of 200 +/- 20 A and 400 +/- 30 A. Diffraction patterns of electron micrographs of these sheets stained with uranyl acetate contain features to 42 A resolution.

Geobacillus stearothermophilus↗
PubMed1986

Characterization of single crystals of the large ribosomal particles from Bacillus stearothermophilus.

Single, three-dimensional crystals of the 50 S ribosomal subunit from Bacillus stearothermophilus (strain NCA) have been characterized using a synchrotron X-ray source. The crystals are orthorhombic with unit cell dimensions: a = 350 A, b = 670 A, c = 905 A, and contain at least one 2-fold screw axis. With cooling to -2 degrees C, the large crystals (1.0 mm X 0.2 mm X 0.1 mm) diffract to 15 to 18 A resolution and are stable in the synchrotron beam for several hours. Despite the large cell dimensions, the reflections are readily resolved when the X-ray diffraction patterns are densitometered with a 25 microns faster.

Crystallization↗
PubMed1983

A compact three-dimensional crystal form of the large ribosomal subunit from Bacillus stearothermophilus.

A new form of well-ordered three-dimensional crystals of intact 50 S ribosomal subunits from Bacillus stearothermophilus have been obtained. Electron micrographs of positively stained sections of these crystals revealed that the ribosomal particles are packed closely. The cell parameters have been determined. Representative electron micrographs and their computed contoured filtered images are shown.

Crystallization↗
PubMed1981

Purification and characterization of demolybdo nitrate reductase (NADH-cytochrome c oxidoreductase) of Chlorella vulgaris.

Chlorella vulgaris was cultured on an ammonia-mineral salts medium until the nitrate reductase content reached a minimal level. These ammonia-grown cells were then induced by nitrate in the absence of molybdenum and of tungsten. A demolybdo nitrate reductase developed and reached high levels. This protein contained very little nitrate-reducing capacity, but had the full cytochrome c-reducing capacity of normal nitrate reductase. It was purified to homogeneity by the same procedures previously developed for the purification of nitrate reductase. The purified enzyme contained 1 molecule of heme and 1 molecule of FAD/subunit, but no detectable molybdenum or tungsten. This cytochrome c reductase was completely inhibited by antibodies raised against purified nitrate reductase of Chlorella. Mixtures prepared from normal nitrate reductase and the demolybdoenzyme could not be resolved by disc gel electrophoresis or by centrifugation in a density gradient. By a two-step enzyme induction (1, incubation with nitrate in absence of Mo; 2, incubation with Mo in absence of nitrate) the process of nitrate reductase synthesis could be cleanly separated from growth into two steps: Step 1, induction of cytochrome c reductase, was completely inhibited by cycloheximide. Step 2 was unaffected by cycloheximide, and most of the nitrate reductase synthesized accumulated in the form of the reversibly inactivated HCN complex of the enzyme.

Chlorella↗
PubMed1980

The formation of hydrogen cyanide from histidine in the presence of amino acid oxidase and peroxidase.

Conditions were sought to increase the yield of HCN from L-histidine incubated with L-amino acid oxidase (L-amino acid:oxygen oxidoreductase (deaminating), EC 1.4.3.2) from snake venom, and horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). Small amounts of histidine and high buffer concentrations favored high HCN yields, which reached a maximum of 72%. Imidazole 4-aldehyde and imidazole 4-carboxylic acid were identified among the reaction products, together with CO2, NH3, H2O2 and imidazole acetic acid. The CO2 formed was equal to the histidine oxidized, and to the sum of NH3 plus HCN formed. The production of HCN was associated with an increased O2 uptake, which was established from the beginning of the reaction, with no apparent lag and ranged from 1.2 to 1.6 mumol extra O2 taken up/mumol HCN formed. The system was inhibited by catalase, but added superoxide dismutase caused a small stimulation of both HCN production and O2 consumption, and a larger stimulation of H2O2 accumulation. Added hydroxylamine was cooxidized to nitrite in an amount equimolar with the HCN formed. This nitrite formation was inhibited by superoxide dismutase. The facts could be interpreted in terms of superoxide anion formation during the HCN-producing reaction. cytochrome c, heme, or ferricyanide could be substituted for peroxidase, but were less effective. The initial rates of HCN formation from phenylalanine, tyrosine and tryptophan were higher, but the eventual yields of HCN from these amino acids were lower than those from histidine.

Amino Acid Oxidoreductases↗