Site-specific integration and excision of pMEA100 in Nocardia mediterranei

Summary Nocardia mediterranei strain LBG A3136 contains the 23.7 kb element pMEA100 in a chromosomally integrated form as well as in the free state (Moretti et al. 1985). The integrated form of this element can be excised precisely from the Nocardia chromosome without any accompanying rearrangements in flanking chromosomal DNA. After transfer into plasmid-free mutant strains, pMEA100 reintegrates site specifically into its original chromosomal locus. The exact mapping of the pMEA100 integration site was accomplished by restriction analysis and DNA sequencing. The attachment site of pMEA100, the junctions of its integrated form and plasmid-free chromosomal DNA of N. mediterranei contain an identical 47 bp long sequence which is probably required for site-specific recombination connected with integration and excision of pMEA100. Only one such sequence was found in the chromosome of pMEA100-free N. mediterranei derivatives as suggested by the single integration locus.     

Patch-clamp study of isolated taste receptor cells of the frog

Summary Taste discs were dissected from the tongue ofR. ridibunda and their cells dissociated by a collagenase/low Ca/mechanical agitation protocol. The resulting cell suspension contained globular epithelial cells and, in smaller number, taste receptor cells. These were identified by staining properties and by their preserved apical process, the tip of which often remained attached to an epithelial (associated) cell. When the patch pipette contained 110mm KCl and the cells were superfused with NaCl Ringer's during whole-cell recording, the mean zero-current potential of 22 taste receptor cells was –65.2 mV and the slope resistance 150 to 750 M. Pulse-depolarization from a holding voltage of –80 mV activated a transient TTX-blockable inward Na current. Activation became noticeable at –25 mV and was half-maximal at –8 mV. Steady-state inactivation was half-maximal at –67 mV and complete at –50 mV. Peak Na current averaged –0.5 nA/cell. The Ca-ionophore A23187 shifted the activation and inactivation curve to more negative voltages. Similar shifts occurred when the pipette Ca was raised. External Ni (5mm) shifted the activation curve towards positive voltages by 10 mV. Pulse depolarization also activated outward K currents. Activation was slower than that of Na current and inactivation slower still. External TEA (7.5mm) and 4-aminopyridine (1mm) did not block, but 5mm Ba blocked the K currents. K-tail currents were seen on termination of depolarizing voltage pulses. A23187 shifted theI _K(V)-curve to more negative voltages. Action potentials were recorded when passing pulses of depolarizing outward current. Of the frog gustatory stimulants, 10mm Ca caused a reversible 5-to 10-mV depolarization in the current-clamp mode. Quinine (0.1mm, bitter) produced a reversible depolarization accompanied by a full block of Na current and, with slower time-course, a partial block of K currents. Cyclic AMP (5mm in the external solution or 0.5 m in the pipette) caused reversible depolarization (to –40 to –20 mV) due to partial blockage of K currents, but only if ATP was added to the pipette solution. Similar responses were elicited by stimulating the adenylate cyclase with forskolin. Blockage of cAMP-phosphodiesterase enhanced the response to cAMP. These results suggest that cAMP may be one of the cytosolic messengers in taste receptor cells. Replacement of ATP by AMP-PNP in the pipette abolished the depolarizing response to cAMP. Inclusion of ATP--S in the pipette caused slow depolarization to –40 to –20 mV, due to partial blockage of K currents. Subsequently, cAMP was without effect. The remaining K currents were blockable by Ba. These results suggest that cAMP initiates phosphorylation of one set of K channels to a nonconducting conformation.     

Molecular studies of the neuronal nicotinic acetylcholine receptor family

Nicotinic acetylcholine receptors on neurons are part of a gene family that includes nicotinic acetylcholine receptors on skeletal muscles and neuronal alpha bungarotoxin-binding proteins that in many species, unlike receptors, do not have an acetylcholine-regulated cation channel. This gene superfamily of ligand-gated receptors also includes receptors for glycine and gamma-aminobutyric acid. Rapid progress on neuronal nicotinic receptors has recently been possible using monoclonal antibodies as probes for receptor proteins and cDNAs as probes for receptor genes. These studies are the primary focus of this review, although other aspects of these receptors are also considered. In birds and mammals, there are subtypes of neuronal nicotinic receptors. All of these receptors differ from nicotinic receptors of muscle pharmacologically (none bind alpha bungarotoxin, and some have very high affinity for nicotine), structurally (having only two types of subunits rather than four), and, in some cases, in functional role (some are located presynaptically). However, there are amino acid sequence homologies between the subunits of these receptors that suggest the location of important functional domains. Sequence homologies also suggest that the subunits of the proteins of this family all evolved from a common ancestral protein subunit. The ligand-gated ion channel characteristic of this superfamily is formed from multiple copies of homologous subunits. Conserved domains responsible for strong stereospecific association of the subunits are probably a fundamental organizing principle of the superfamily. Whereas the structure of muscle-type nicotinic receptors appears to have been established by the time of elasmobranchs and has evolved quite conservatively since then, the evolution of neuronal-type nicotinic receptors appears to be in more rapid flux. Certainly, the studies of these receptors are in rapid flux, with the availability of monoclonal antibody probes for localizing, purifying, and characterizing the proteins, and cDNA probes for determining sequences, localizing mRNAs, expressing functional receptors, and studying genetic regulation. The role of nicotinic receptors in neuromuscular transmission is well understood, but the role of nicotinic receptors in brain function is not. The current deluge of data using antibodies and cDNAs is beginning to come together nicely to describe the structure of these receptors. Soon, these techniques may combine with others to better reveal the functional roles of neuronal nicotinic receptors.     

Biochemical characterization of chromatin fractions isolated from induced and uninduced friend erythroleukemia cells

Summary Chromatin fractions from Friend erythroleukemia cells after induction of differentiation by dimethylsulfoxide (DMSO) were compared in their biochemical characteristics to fractions from uninduced cells. Fractions were prepared by extracting chromatin from nuclei after mild micrococcal nuclease treatment with increasing concentrations of NaCl according to Sanders [1]. This procedure has been found to release chromatin containing hyperacetylated histones preferentially [2]. The fractions obtained by this procedure were analysed in respect to the amount of chromatin released, the amount of histone H1, the degree of acetylation of histone H4, the presence of non-histone proteins and the concentration of transcribed and non-transcribed sequences. It was found that the fractions differ in the amount of histone H1 present, in several non-histone proteins and in the acetylation of histonie H4, regardless whether induced or uninduced cells were analysed. The distribution of transcribed sequences versus non-transcribed sequences among the fractions was the same, demonstrating that this fractionation procedure, although leading to fractions with biochemical differences, is not able to discriminate functional states of chromatin and that the biochemical characteristics of the fractions may be common to both, active as well as inactive states of chromatin.     

Transformation of Claviceps purpurea using a bleomycin resistance gene

Summary To develop a DNA-mediated transformation system for Claviceps purpurea a vector was constructed using a bleomycin-resistance gene (bleo ^R) fused in frame to the Aspergillus nidulans trp C promoter as a dominant selection marker. The construct was shown to be functional in Aspergillus nidulans and Aspergillus niger and used to transform a wild strain of Claviceps purpurea. Transformats were obtained at low frequencies; they were shown to contain transforming DNA integrated into the chromosomal DNA, probably in multimeric copies and at multiple sites. Combined Southern, Northern and resistance level analysis indicate that the A. nidulans promoter is functional in C. purpurea.Dedicated to Professor Dr. Dr. h. c. K. Esser on the occasion of his 65^th birthday     

The electrochemical proton potential generated by the sulphur respiration of Wolinella succinogenes

Wolinella succinogenes grown on formate and elemental sulphur was found to use the polysulphide derivatives 2,2-tetrathiobispropionate (R₂S₄) or pentathionate (S₅O ₆ ⁼ ) as acceptors for formate oxidation. The specific activities of formate oxidation with these acceptors were similar to those with elemental sulphur. The main reaction products of R₂S₄ reduction were 2,2-dithiobispropionate (R₂S₂) and sulphide. Pentathionate was converted to thiosulphate and some elemental sulphur. The electrochemical proton potential across the cytoplasmic membrane of the bacterium was measured in the steady state of electron transport from formate to R₂S₄. The electrical proportion () of the determined through the distribution of labeled tetraphenylphosphonium cation was obtained as 0.17 Volt. The was zero, when a protonophore was present. The pH-difference across the membrane was negligible. Thus the generated by sulphur respiration is close to that measured earlier with fumarate as the terminal acceptor of electron transport.Abbreviations DMO 5,5-dimethyloxazolidine-2,4-dione - R₂S_n (n=2–5) 2,2-polythiobispropionate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazol - TPP tetraphenylphosphonium cation     

Die Verbreitung des Weidensperlings (Passer hispaniolensis) in Marokko

Zusammenfassung In Marokko fällt die südliche Verbreitungsgrenze des Weidensperlings wahrscheinlich mit dem Nordrand des Antiatlas zusammen. Aus den Oasen der ehemaligen Spanischen Sahara liegen keine Beobachtungen vor. Aus dem Raum Oujda sind die östlichsten Populationen, die ständig zwischen Marokko und Algerien migrieren, bekannt. Landnutzung und Klima prägen entscheidend die Dispersion des Weidensperlings. Als euryöke Art ist er in der Lage, das reichhaltige Angebot an Schlaf- und Brutplätzen sowie an Nahrung und Wasser in den ackerbaulichen Nutzflächen zu verwerten. Auffallend ist, daß die Hauptverbreitungszonen mit den großen ackerbaulichen Nutzflächen zusammenfallen. Während die agrarischen Nutzflächen sich in den letzten Jahrzehnten im Zuge der Modernisierung der Landwirtschaft rapide änderten, und das erklärte Ziel für das Jahr 2000 in einer bewässerten Fläche von einer Million Hektar besteht, gingen die ursprünglichen Habitate des Weidensperlings, wie die Bereiche der mauretanischen Wüstensteppengebiete, drastisch zurück. Ferner konnte im Raum Marrakech eine Abnahme der Niederschläge beobachtet werden, die das für die Weidensperlinge verfügbare Nahrungsangebot entscheidend beeinflußt und somit die Populationsdynamik steuert.

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On the distribution of the Spanish Sparrow (Passer hispaniolensis) in Morocco

Summary In Morocco, the southernmost border of the distribution of the Spanish Sparrow coincides probably with the North-Antiatlas. From the oasis of the former Spanish Sahara don't exist any observations. In the region of Oujda the easternmost populations are found which are migrating permanently between Morocco and Algeria. The euryoecous species is able to use a high variability of different roosts, breeding sites, resources of food and water in the agricultural areas. A high overlap of the main distribution areas with the large agricultural regions is obvious. While the cultivated areas have been changed heavily during the modernisation periode of the Moroccan agriculture the original habitats of the Spanish Sparrow (i. e. the parts in the Mauretanian desert-steppe regions) disappeared dramatically. The goal of the year 2000 lies in an irrigated scheme of 1.000.000 ha. Moreover, a decrease of the precipitation in the region of Marrakech was observed, which has a great influence on the food available for the species and which therefore controls the population dynamics.

     

Search for polythionates in cultures of Chromatium vinosum after sulfide incubation

Cultures of Chromatium vinosum, devoid of sulfur globules, were supplemented with sulfide and incubated under anoxic conditions in the light. The concentrations of sulfide, polysulfides, thiosulfate, polythionates and elemental sulfur (sulfur rings) were monitored for 3 days by ion-chromatography and reversed-phase HPLC. While sulfide disappeared rapidly, thiosulfate and elemental sulfur (S₆, S₇ S₈ rings) were formed. After sulfide depletion, the concentration of thiosulfate decreased fairly rapidly, but elemental sulfur was oxidized very slowly to sulfate. Neither polysulfides (S _x ^2– ), polythionates (S_nO ₆ ^2– , n=4–6), nor other polysulfur compounds could be detected, which is in accordance with the fact that sulfide-grown cells were able to oxidize polysulfide without lag. The nature of the intracellular sulfur globules is discussed.     

Soils contain two different activities for oxidation of hydrogen

Abstract Hydrogen oxidation rates were measured in a neutral compost soil and an acidic sandy loam at H₂ mixing ratios of 0.01 to 5000 ppmv. The kinetics were biphasic showing two different K _m values for H₂, one at about 10–40 nM dissolved H₂, the other at about 1.2–1.4 μM H₂. The low- K _m activity was less sensitive to chloroform fumigation than the high- K _m activity. If sterile soil was amended with Paracoccus denitrificans or a H₂-oxidizing strain isolated from compost soil, it exhibited only a high- K _m (0.7–0.9 μM) activity. It also failed to utilize H₂ mixing ratios below a threshold of 1.6–3.0 ppmv H₂ (160–300 mPa). A similar result was obtained when fresh soil samples were suspended in water, and H₂ oxidation was determined from the decrease of dissolved H₂. However, H₂ was again utilized to mixing ratios lower than 0.05 ppmv, if the supernatant of the soil suspension or the settled soil particles were dried onto sterile soil or purified quarz sand. Obviously, soils contain two different activities for oxidation of H₂: (1) a high- K _m, high-threshold activity which apparently is due to aerobic H₂-oxidizing bacteria, and (2) a low- K _m, low-threshold activity whose origin is unknown but presumably is due to soil enzymes.