Computer-Assisted Image Analysis of Plant Growth, Thigmomorphogenesis, and Gravitropism

A nonintrusive auxonometric system, based on the DARWIN image processor (Telewski et al. 1983 Plant Physiol 72: 177-181), is described and demonstrated in the analysis of gravitropism and thigmomorphogenesis in corn seedlings (Zea mays). Using this system, growth and bending of regularly shaped plants or organs can be quickly and accurately measured without, in any way, interfering with the plant. Furthermore, the growth and bending curves are automatically plotted. Thigmomorphogenesis in the aerial part of corn seedlings involves growth promotion at a low force load and growth retardation at higher force loads. The time courses of the two kinds of response are somewhat different, with retardation occurring immeditely after mechanical perturbation and growth promotion taking somewhat longer to begin. Gravitropic experiments show that when dark-grown corn seedlings are placed on their side in the light, the resulting curvature is due to two consecutive morphological mechanisms. In the first instance, lasting for about 15 minutes, the elongation of the bottom edge of the plant accelerates, while the elongation of the top edge remains constant. After that, for the next 1.75 hours, the elongation of the top edge decelerates and stops while that of the bottom edge remains constant at the increased rate for most of the period. The measurements taken from both experiments at relatively high resolution (0.08-0.1 millimeter) show that the growth curves are not smooth but show many small irregularities which may or may not involve micronutations.     

Isolation and characterization of a blue copper protein from Thiobacillus versutus

The blue copper protein induced during growth of Thiobacillus versutus on methylamine was purified and characterized. It is an acidic protein (isoelectric point 4.7), contains one Cu2+ ion/enzyme molecule, is a monomeric protein (molecular mass about 14 kDa), has a maximum in its absorption spectrum at 596 nm (molar absorption coefficient 3.9 X 10(3) M-1 cm-1), shows an axial type-I electron paramagnetic resonance spectrum (g parallel = 2.239, g perpendicular = 2.046 and A parallel = 5.6 mT) and has a redox potential (Eo) of + 260 mV. In view of these properties and in view of the fact that the protein is active as an electron carrier between methylamine dehydrogenase and cytochrome c, it is concluded that it is similar to the amicyanins isolated from Methylomonas sp. strain J and Pseudomonas sp. strain AM 1.     

Variation in alcohol dehydrogenase activity in vitro in flies from natural populations ofDrosophila melanogaster

Alcohol dehydrogenase (ADH) activity variation in male flies taken directly from seven natural populations ofDrosophila melanogaster is largely accounted for by segregation of alleles at theAdh structural gene locus. There was little overlap in the ADH activities ofAdh ^F andAdh ^s homozygotes. Body weights varied only slightly betweenAdh genotypes and contributed little to ADH variation. Between and within population variation in ADH activity and ADH protein in flies in the wild is mainly due to the relative frequencies ofAdh ^F andAdh ^s.     

Green-backed herons (Butorides striatus) possibly using a lure and using apparent bait

Zusammenfassung In Burkina Faso ließ ein Mangrovereiher offensichtlich gezielt eine Asclepiadaceen-Blüte aus 20 cm Höhe auf die Wasseroberfläche fallen und verharrte danach einige Sekunden mit halb-gestrecktem Hals. Bei einer weiteren Beobachtung in Niger plazierte ein Mangrovereiher einen kleinen Gegenstand auf der Wasseroberfläche. Bevor der Gegenstand mit dem Wind außer Reichweite trieb, holte ihn der Reiher und legte ihn auf der Luvseite wieder auf der Wasseroberfläche ab. Der Vorgang wiederholte sich mehrere Male, dabei gelang es dem Reiher, einen Fisch zu erbeuten, der allerdings wieder entkam. Anderntags setzte an derselben Stelle ein Mangrovereiher in gleicher Weise offensichtlich einen Käfer ein. Ähnliche Beobachtungen werden kurz diskutiert.     

Complementation between uncF alleles affecting assembly of the F1F0-ATPase complex of Escherichia coli.

A mutant affected in the b subunit (coded by the uncF gene) of the F1F0-ATPase in Escherichia coli was isolated by a localized mutagenesis procedure in which a plasmid carrying the unc genes was mutagenized in vivo. The biochemical properties of cells carrying the uncF515 allele were examined in a strain carrying the allele on a multicopy plasmid and a mutator-induced polar unc mutation on the chromosome. The strain carrying the mutant unc allele was uncoupled with respect to oxidative phosphorylation. Membrane-bound ATPase activity was very low or absent, and membranes were somewhat proton permeable. It was concluded that the F0 sector was assembled. Determination of the DNA sequence of the uncF515 allele showed it differed from wild type in that a G----A substitution occurred at position 392, resulting in glycine being replaced by aspartate at position 131. Genetic complementation tests indicated that the uncF515 allele complemented the uncF476 allele (Gly 9----Asp). Two-dimensional gel electrophoresis of membrane preparations indicated that the uncF515 and uncF476 alleles interrupted assembly of the F1F0-ATPase at different stages.     

A selenomethionine-containing azurin from an auxotroph of Pseudomonas aeruginosa

The production and spectroscopic properties of an L-selenomethionine-containing homolog of Pseudomonas aeruginosa azurin are described. The amino acid substitution was carried out by developing an L-methionine-dependent bacterial strain from a fully functional ATCC culture. Uptake studies monitored using L-[75Se]methionine indicated that L-selenomethionine was incorporated into the protein synthetic pathway of Pseudomonas bacteria in a manner analogous to L-methionine. Several batches of bacteria were grown, and one sample of isolated and purified selenoazurin (azurin in which methionine was substituted by selenomethionine) was found (by neutron activation analysis) to contain 5.2 +/- 0.8 seleniums/copper. Correspondingly, a residual 0.35 methionines, relative to 6.0 in the native protein, were found by amino acid analysis in this azurin sample. The redox potential and extinction coefficient of this selenoazurin were found to be 333 +/- 1 mV (pH 7.0, I = 0.22) and 5855 +/- 160 M-1 cm-1 at 626 +/- 1 nm, respectively. Visible electronic, CD, and EPR spectra are reported and Gaussian curve fitting to the former spectrum allowed assignment of the selenomethionine Se----Cu(II) transition to a band found at 18034 cm-1, based upon an observed 450 cm-1 shift to the red from the analogous band position in the native protein. The data are consistent with a relatively more covalent copper site stabilizing the reduced, Cu(I), form in the selenoprotein. A role for the methionine as a modulator of the blue copper site redox potential by metal----ligand back bonding from Cu(I) is discussed in terms of a ligand sphere which limits the valence change at copper to much less than 1 during a redox cycle.     

Cytoplasmic methionyl-tRNA synthetase from Bakers' yeast. A monomer with a post-translationally modified N terminus

Methionyl-tRNA synthetase has been purified from a yeast strain carrying the MES1 structural gene on a high copy number plasmid (pFL1). The purified enzyme is a monomer of Mr = 85,000 in contrast to its counterpart from Escherichia coli which is a dimer made up of identical subunits (Mr = 76,000; Dardel, F., Fayat, G., and Blanquet, S. (1984) J. Bacteriol. 160, 1115-1122). The yeast enzyme was not amenable to Edman's degradation indicating a blocked NH2 terminus. Its primary structure as derived from the DNA sequence (Walter, P., Gangloff, J., Bonnet, J., Boulanger, Y., Ebel, J.P., and Fasiolo, F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2437-2441) has been confirmed using the fast atom bombardment-mass spectrometric method. This method was applied to tryptic digests of the carboxymethylated enzyme and the corresponding data provided extensive coverage of the translated DNA sequence, thus confirming its correctness. The ambiguity concerning which of the three NH2-terminally located methionine codons is the initiation codon was easily resolved from peptides identified in this region. It was possible to show that the first methionine had been removed and that the new NH2 terminus, serine, had been acetylated. A comparison between the yeast and E. coli sequences shows that the former has an N-terminal extension of about 200 residues as compared to the latter. It also lacks the C-terminal domain which is responsible for the dimerization of the E. coli methionyl-tRNA synthetase.     

Improved Method for Isolating Synaptosomes from 11 Regions of One Rat Brain: Electron Microscopic and Biochemical Characterization and Use in the Study of Drug Effects on Nerve Terminal γ-Aminobutyric Acid in Vivo

A procedure is described for the rapid preparation of nerve ending particles (synaptosomes) from 11 regions of one rat brain. The synaptosomal fractions have been characterized by electron microscopy and determination of four marker enzymes, i.e., glutamate decarboxylase (GAD), acetylcholinesterase, succinate dehydrogenase, and glycerol 3-phosphate dehydrogenase. Comparison with a much lengthier standard (Ficoll-sucrose) preparation showed that the synaptosomal yield of the new procedure was substantially better as judged by both morphological evaluation and protein recovery. The improved synaptosome preparation was used for determination of regional gamma-aminobutyric acid (GABA) levels in synaptosomal fractions. The postmortem increase in GABA level during removal and dissection of brain tissue and homogenization and fractionation procedures could be minimized by rapid processing of the tissue at low temperatures and inclusion of the GAD inhibitor 3-mercaptopropionic acid (3-MP; 1 mM) in the homogenizing medium. The addition of GABA (0.2 mM) to the homogenizing medium did not alter the GABA levels in the synaptosomes, indicating that no significant redistribution of GABA occurred during subcellular fractionation in sodium-free media. Synaptosomal GABA levels determined in the 11 rat brain areas showed the same regional distribution as the GABA-synthesizing enzyme GAD. On the basis of these findings, it was suggested that the synaptosome preparation could be used to evaluate the in vivo effects of drugs on nerve terminal GABA. Treatment of rats with a convulsant dose of 3-MP (50 mg/kg i.p.) 3 min before decapitation significantly lowered synaptosomal GABA levels in olfactory bulb, hippocampus, thalamus, tectum, and cerebellum. The 3-MP-induced seizures and reduction of GABA levels could be prevented by administration of valproic acid (200 mg/kg i.p.) 15 min before the 3-MP injection. The data indicate that the improved synaptosome preparation offers a convenient method of preparing highly purified synaptosomes from a large number of small tissue samples and can provide useful information on the in vivo effects of drugs on regional GABA levels in nerve terminals.     

Molecular cloning of a pea mRNA encoding an early light induced,nuclear coded chloroplast protein

Summary cDNA clones were isolated for a chloroplast protein, the mRNA of which is induced to maximum levels within 2–4 h after onset of illumination in five day old, etiolated pea seedlings. The cDNA library was constructed from poly(A)⁺-mRNA which was isolated from 4 h illuminated seedlings. The extremely short induction period of the early light induced protein(ELIP)-mRNA established the basis of our screening procedure. Colony hybridization experiments were performed with³²P-labelled cDNA probes, synthesized from RNA of seedlings which had been exposed to different programs of illumination. Plasmid DNAs were isolated from colonies showing strong hybridization signals exclusively with cDNA corresponding to the 4 h-mRNA. Hybrid released translation of preselected plasmids p 17/C2 and p17/C4 revealed a peptide of M_r 24 000. After posttranslational importin vitro, the processed product of M_r 17 000 appears in the chloroplast. Using these clones, the expression of the ELIP-mRNA was investigated by DOT-hybridization. The ELIP-mRNA reaches maximum levels within 2–4 hours after onset of illumination. Our results correspond precisely to thein vivo characteristics and indicate positive identification of the sought clones.