Preparation and properties of recombinant DNA derived tobacco mosaic virus coat protein

Recombinant DNA derived tobacco mosaic virus (vulgare strain) coat protein (r-TMVP) was obtained by cloning and expression in Escherichia coli and was purified by column chromatography, self-assembly polymerization, and precipitation. SDS-PAGE, amino terminal sequencing, and immunoblotting with polyclonal antibodies raised against TMVP confirmed the identify and purity of the recombinant protein. Isoelectric focusing in 8 M urea and fast atom bombardment mass spectrometry demonstrated that the r-TMVP is not acetylated at the amino terminus, unlike the wild-type protein isolated from the tobacco plant derived virus. The characterization of r-TMVP with regard to its self-assembly properties revealed reversible endothermic polymerization as studied by analytical ultracentrifugation, circular dichroism, and electron microscopy. However, the details of the assembly process differed from those of the wild-type protein. At neutral pH, low ionic strength, and 20 degrees C, TMVP forms a 20S two-turn helical rod that acts as a nucleus for further assembly with RNA and additional TMVP to form TMV. Under more acidic conditions, this 20S structure also acts as a nucleus for protein self-assembly to form viruslike RNA-free rods. The r-TMVP that is not acetylated carries an extra positive charge at the amino terminus and does not appear to form the 20S nucleus. Instead, it forms a 28S four-layer structure, which resembles in size and structure the dimer of the bilayer disk formed by the wild-type protein at pH 8.0, high ionic strength, and 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the S'-subsite specificity of V8 proteinase via acyl transfer to added nucleophiles

The S'-subsite specificity of endoproteinase Glu-C (V8 proteinase) was studied by acyl transfer reactions using Z-Glu-OMe as acyl donor and a series of amino acid- and peptide-derived nucleophiles. The partition constant, which characterizes specificity, was determined by a method based on the integrated rate equation. V8 proteinase prefers amino acid residues with hydrophobic side chains in the P'1 position. Di- and tripeptide amides are more efficient nucleophilic amino components than amino acid amides.     

Evaluation of experimental combined toxicity by use of dose-frequency curves: comparison with theoretical additivity as well as independence

Dose-frequency curves of toxic effects of a substance A were evaluated in the absence and in the presence of a fixed dose of a second substance B. Data were fitted by the curve-fitting program ALLFIT. Observed combined frequencies of A + B were compared statistically with the expected frequencies of additivity and (or) independence by the phi 2-square goodness-of-fit test. The theoretical dose-frequency curves expected for an additive response were obtained by a solely graphical procedure and the theoretical curves for independent effects were calculated from the effects of B and A at certain doses. In rotarod tests with trained mice, the combined deteriorating effect of ethanol and benzodiazepines were significantly over-additive. However, their lethal interaction appeared underadditive in mice. The lethal underadditive interaction of ethanol and phencyclidine (PCP) can be ascribed largely to independent actions of these compounds. Loss of righting reflex was additively enhanced by PCP, whereas PCP overadditively enhanced the effect of ethanol. The insecticidal action of the cholinesterase inhibitors malathion and parathion appeared additive and significantly different from independent interaction. A comparison of results from dose-response curves with isoboles showed good agreement. The method appears as an attractive alternative or as a complementary procedure to the isobolographic analysis. Combination experiments as described can be carried out and evaluated rather simply, with a minimum of expenditure and a maximum of information.     

A bacteriophage P1-encoded modulator protein affects the P1 c1 repression system

Bacteriophage P1 encodes a tripartite immunity system composed of the immC, immI, and immT region. Their basic genetic elements are the c1 repressor of lytic functions, the c4 repressor which negatively regulates antirepressor synthesis, and the bof gene, respectively. The function of the latter will be described here. We have cloned and sequenced the bof gene from P1 wild type and a P1 bof amber mutant. Based on the position of a TAG codon of the bof amber mutant the bof wild type gene was localized. It starts with a TTG codon, comprises 82 codons, and is preceded by a promoter structure. The bof protein (Mr = 7500) was overproduced in Escherichia coli from a bof recombinant plasmid and was purified to near homogeneity. The N-terminal amino acids predicted from the DNA sequence of the bof gene were confirmed by sequence analysis of the bof protein. Using a DNA mobility shift assay, we show that bof protein enhances the binding of c1 repressor to the operator of the c1 gene. In accordance with this result, in transformants of Escherichia coli, containing both a bof- and a c1-encoding plasmid, c1 expression is down-regulated. We conclude that bof acts as a modulator protein in the repression of a multitude of c1-controlled operators in the P1 genome.     

Aggression by the damselfish Dascyllus aruanus L. Towards conspecifics and heterospecifics

Field experiments were conducted to demonstrate species recognition in Dascyllus aruanus. Aggression was used as a criterion for recognition. Free-living fish were presented with live stimuli in a choice situation where visual cues could be isolated from other cues. The stimuli were conspecific and heterospecific fish. Practically all the responses were aggressive. Aggression towards D. aruanus and Dascyllus marginatus was similar in its intensity, and higher than towards Dascyllus trimaculatus. Aggression towards Amphiprion bicinctus was negligible. Elimination of all but visual cues resulted in a decline in attack rate without changing attack preference. The high level of interspecific aggression limits the use of aggression as an indicator of species recognition under the presented experimental situation.     

Die Variabilität vegetativer und generativer Flechtenstrukturen und ihre Bedeutung für die Systematik

Die Entstehung von Stützleisten in den Thalli von Cladonia chlorophaea und Cetraria islandica, sowie die Entwicklung sekundärer Soredien im Inneren der Podetien von Cladonia chlorophaea werden beschrieben. Gemeinsam mit anderen Entwicklungsvorgängen von Flechten zeigen die Beobachtungen eine sehr geringe Determination der Flechtengewebe. Die Struktur wird weitgehend durch die Funktion bestimmt, während gleichzeitig bestimmte ontogenetische Abläufe unveränderbar sind. Die Bedeutung dieser Erscheinungen für die Systematik wird diskutiert.     

Structural preferences for the binding of chromium nucleotides by beef heart mitochondrial ATPase

The mono- and bidentate forms of adenosine 5'-diphosphate, chromium (III) salt (CrADP) were separated using Sephadex G-10 column chromatography. The isomeric purity of the two forms was monitored using high voltage electrophoresis and column chromatography. The same techniques were employed to assess the purity of the mono-, bi-, and tridentate forms of adenosine 5'-triphosphate, chromium (III) salt (CrATP). Distinct differences in the interaction of beef heart mitochondrial ATPase with the various isomers of chromium nucleotides were seen in kinetic studies. Monodentate CrADP was a competitive inhibitor of the ATP hydrolysis activity of both purified ATPase and submitochondrial particles. However, when ITPase activity was examined, noncompetitive inhibition was observed. The bidentate isomer of CrADP did not affect ATPase activity. Enzymatic synthesis of the transition state analog of ATP synthesis and hydrolysis, Pi-CrADP occurred exclusively with the monodentate isomer of CrADP. It was also found that only the mono- and tridentate forms of CrATP were potent inhibitors of ATP hydrolysis by beef heart mitochondrial ATPase. These results are discussed in terms of possible ATP synthesis and hydrolysis mechanisms.