Isolation and characterization of the dut gene of Escherichia coli. II. Restriction enzyme mapping and analysis of polypeptide products

Restriction endonuclease mapping of previously constructed dut plasmids has been carried out using the enzymes PvuI, PvuII and SacI. Various dut plasmids were also tested in the "maxicell" protein-synthesizing system. They all show two protein bands in common, one of Mr 16000 in agreement with the size previously reported for the purified dUTPase subunit (Shlomai and Kornberg, 1978). With the information obtained the structural gene for dUTPase can be assigned to a 950-bp SacI-PvuII fragment of the E. coli genome. Studies, described in the preceding paper, on the overproduction of dUTPase by bacterial strains carrying different dut plasmids strongly suggest that the dut gene is transcribed in the direction from the SacI site towards the PvuII site and that the SacI site is located within the dut control region. The second protein band observed in the "maxicell" experiments has an Mr of 23500. Its identity is unknown but it may represent a precursor of dUTPase or the product of a separate gene located between dut and pyrE.     

High surface hydrophobicity ofStaphylococcus aureus as revealed by hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) on Octyl Sepharose^R in a column procedure was used to compare the relative surface hydrophobicity ofStaphylococcus aureus reference strains, protein A-negative mutants, and strains isolated from bovine mastitis. High protein A-producing strains (Cowan 1 and clinical isolate SA 17970) showed a higher relative surface hydrophobicity than did strains producing a low amount of protein A. One encapsulatedS. aureus strain (Smith diffuse) did not bind to the gel, while an unencapsulated variant showed binding properties similar to weak protein A-producing strains. Studies onS. aureus strains isolated from bovine mastitis revealed a good correlation between adsorption to Octyl Sepharose and the production of protein A. Results indicate that protein A and probably other surface proteins such as fibronectin-binding protein contribute to the high relative surface hydrophobicity ofS. aureus.     

Gene fusion vectors based on the gene for staphylococcal protein A

Two plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusion, have been constructed. These vectors will allow fusion of any gene to the protein A gene, thus giving hybrid proteins which can be purified, in a one-step procedure, by IgG affinity chromatography. As an example of the practical use of such vectors, the protein A gene has been fused to the lacZ gene of Escherichia coli. E. coli strains containing such plasmids produce hybrid proteins with both IgG binding and β-galactosidase activities. The hybrid protein(s) can be immobilized on IgG-Sepharose by its protein A moiety with high efficiency without losing its enzymatic activity and they can be eluted from the column by competitive elution with pure protein A. The fused protein(s) also binds to IgG-coated microtiter wells which means that the in vivo product can be used as an enzyme conjugate in ELISA tests.     

Mikromorphologie und Orientierungsmuster epicuticularer Wachs-Kristalloide: Ein neues systematisches Merkmal bei Monokotylen

Based on SEM examinations of some 1600 species of monocotyledons out of 61 families the characteristics of their epicuticular waxes are described in this preliminary paper and illustrated by 20 SEM micrographs. Four basic types of waxes are recognized. Two of them occur throughout the monocotyledonous and dicotyledonous orders of angiosperms; two others are of high systematic significance and restricted to particular orders of the monocotyledons: The “Convallaria-Type” is characterized by fine wax platelets with parallel orientation, forming a pattern around the stomata reminiscent of electromagnetic field lines. This type is restricted to the three liliiflorous ordersAsparagales, Liliales, andBurmanniales. The “Strelitzia-Type” is characterized by massive compound wax rodlets; it is restricted to the superordersAreciflorae, Commeliniflorae, Zingiberiflorae, plus the two liliiflorous ordersBromeliales andVelloziales. The data are compared with the classifications ofDahlgren & Clifford (1982); they tentatively indicate the exclusion ofBromeliales, Velloziales, andTyphales from theLiliiflorae.     

Productivity and temperature biology of two snowbed bryophytes

Summary Chlorophyll distribution within the carpets, CO₂ gas exchange under controlled conditions, and heat resistance of the snowbed bryophyte Anthelia juratzkana (Limpr.) Trev. were investigated. Also the gas-exchange parameters of the co-occurring Polytrichum sexangulare Floercke were assessed. Only the uppermost 4 mm layer of Anthelia carpets contains sufficient pigments for photosynthesis. At light saturation and optimal temperatures (6–11°C) the maximum rates of CO₂ uptake are 0.7 mg CO₂ g^-1dw h^-1 in Anthelia and 1.5 mg CO₂ g^-1dw h^-1 in Polytrichum. Gas exchange reaches light saturation at about 300 E m^-2s^-1 in both species. At +2°C the light compensation point is reached at ca. 10E m^-2s^-1 and increases significantly with increasing temperature. The lower temperature compensation point is reached at-4°C in Anthelia and does not drop much below-5°C in Polytrichum. Anthelia cannot sustain net photosynthesis beyond 30°C and Polytrichum not beyond 32°C. Nine month storage under dark, cold and wet conditions does not affect the photosynthetic capability of Anthelia. As a response, however, the net photosynthesis rate is depressed due to an increase of the respiration rates. Polytrichum sexangulare did not tolerate the storage so well. The heat resistance limit of Anthelia is low (39°C). There is evidence that the distribution of the two bryophytes within snowbed communities is determined by their capability to make use of low light intensities and their low temperature demand for optimal photosynthetic rates. Being resistant to long lasting cold, wet, and dark conditions, Anthelia is particularly adapted to grow in the border zone along permanent snowpatches. Polytrichum is more productive and is therefore capable of competing successfully at sites which are less extreme and therefore accessible for higher plants.     

Studies on tissue cultures of the genus Cinchona L. alkaloid production in cell suspension cultures

Initiation and culture of callus and cell suspensions of Cinchona ledgeriana and C. succirubra as well as the successful isolation and selection of a high-yielding alkaloid-forming strain derived from the leaf rachis of a C. succirubra plant are described. Results of feeding experiments with L-tryptophan using two different culture procedures are presented and discussed. Maximum alkaloid yields of up to 0.9% (based on dry weight) or 6.35 mg/l have been obtained.     

Indole alkaloids from cell suspension cultures of Tabernaemontana divaricata and Tabernanthe iboga

From cell suspension cultures of Tabernaemontana divaricata and Tabernanthe iboga grown under standard conditions, six monoterpenoid indole alkaloids have been isolated and identified. T. divaricata synthesized apparicine, catharanthine, coronaridine, conoflorine, tubotaiwine and vinervine, whereas T. iboga produced tubotaiwine and conoflorine. Both cultures are a reasonable source for conoflorine, which is expected to be a good candidate for studying the mechanism of Aspidosperma type alkaloid formation at the cell-free level.     

The oxidation-reduction kinetics of the reaction of cytochrome c1 with non-physiological redox agents

The kinetics of the oxidation-reduction reactions of cytochrome c₁ with ascorbate, ferricyanide, triphenanthrolinecobalt(III) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) have been examined using the stopped-flow technique. The reduction of ferricytochrome c₁ by ascorbic acid is investigated as a function of pH. It is shown that at neutral and alkaline pH the reduction of the protein is mainly performed by the doubly deprotonated form of ascorbate. From the ionic-strength-dependence studies of the reactions of cytochrome c₁ with ascorbate, ferricyanide and triphenanthrolinecobalt(III), it is demonstrated that the reaction rate is governed by electrostatic interactions. The second-order rate constants for the reaction of cytochrome c₁ with ascorbate, ferricyanide, TMPD and triphenanthrolinecobalt(III) are 1.4·10⁴, 3.2·10³, 3.8·10⁴ and 1.3·10⁸ M^?1·s^?1 (pH 7.0, I = 0, 10°C), respectively. Application of the Debye-Hückel theory to the the ionic-strength-dependence studies of these redox reactions of cytochrome c₁ yielded for ferrocytochrome c₁ and ferricytochrome c₁ a net charge of ?5 and ?4, respectively. The latter value is close to that of ?3 for the oxidized enzyme, calculated from the amino acid sequence of the protein. This implies that not a local charge on the surface of the protein, but the overall net charge of cytochrome c₁ governs the reaction rate with small redox molecules.     

Der Einfluss von CCC auf die Entwicklung des Roggenhalmes

Field trials on the effect of chlorocholinechloride (CCC) on rye plants of the cultivar Danae and of a selected population “WRS” proved that rye principally shows as reaction analogous to wheat. The CCC-induced decrease of stalk length is due to the reduction of elongation growth of the 4th internode. This shortening effect is mainly the result of decreased cell extension and, in the middle internode, additionally of inhibited cell division in longitudinal direction. The shape of internodes is changed under the influence of CCC. Walls of parenchyma cells of CCC-treated plants are thinner and those of sclerenchyma cells are thicker compared with cell walls of control plants.