Role of Heme in Synthesis and Membrane Binding of Succinic Dehydrogenase in Bacillus subtilis

A 5-aminolevulinic acid-requiring mutant of Bacillus subtilis was isolated. When the mutant is shifted from medium containing 5-aminolevulinic acid to medium lacking this growth factor, the bacteria continued to grow at undiminished rate for about three generations. The membranes from these bacteria contained severely reduced amounts of cytochrome. The mutant was used to study the role of heme synthesis on synthesis and membrane binding of succinic dehydrogenase (SDH). The amount of SDH in whole-cell lysates in the soluble cytoplasmic fraction and in membranes was determined by one-dimensional (rocket) immunoelectrophoresis with an SDH-specific antiserum. After heme synthesis was blocked, the relative amount of SDH in the membrane decreased, whereas increasing amounts of SDH antigen were found in the cytoplasm. When heme synthesis was resumed on readdition of 5-aminolevulinic acid, the amount of membrane-bound SDH antigen increased at a much faster rate than net synthesis. During a 3-h growth period without 5-aminolevulinic acid, there was little change in the pattern of membrane proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioactively labeled membranes, as compared to membranes from control cultures. However, both the 65,000-dalton and the 28,000-dalton polypeptides of the SDH complex (L. Hederstedt, E. Holmgren, and L. Rutberg, J. Bacteriol. 138:370-376, 1979) were present in decreasing amounts in membranes from 5-aminolevulinic acid-starved bacteria. From these results we suggest that SDH in B. subtilis is synthesized as a soluble protein and becomes membrane bound only when it attaches to a site in the membrane, (part of) which is a cytochrome of b type.     

The chromatin structure of specific genes: I. Evidence for higher order domains of defined DNA sequence.

When the chromatin of Drosophila is examined by digestion with DNAase I or micrococcal nuclease, no general structural organization above the level of the nucleosome is revealed by the cleavage pattern. In contrast, the DNAase I cleavage pattern of specific regions of the Drosophila chromosome shows discrete bands with sizes ranging from a few kilobase pairs (kb) to more than 20 kb. Visualization of such higher order bands was achieved by the use of the Southern blotting technique. The DNAase I-cleaved fragments were transferred onto a nitrocellulose sheet after size fractionation by gel electrophoresis. Hybridization was then carried out with radioactively labeled cloned fragments of DNA from D. melanogaster. For the five different chromosomal regions examined, each gives a unique pattern of higher order bands on the autoradiogram; the patterns are different for different regions. Restriction enzyme cleavage of the fragments generated indicates that the preferential DNAase I cleavage sites in chromatin are position-specific. The chromosomal regions bounded by preferential DNAase I cleavage sites are referred to as supranucleosomal or higher order domains for purposes of discussion and analysis. The micrococcal nuclease cleavage pattern of chromatin at specific loci was also examined. In the one case studied in detail, this nuclease also cleaves at position-specific sites.     

Effects in vitro of alloxan on the glucose metabolism of mouse pancreatic B-cells

To facilitate detailed studies of the B-cytotoxic action of alloxan we developed a model using isolated pancreatic islets of normal mice. An essential feature of this model is the low temperature employed during exposure to alloxan, which minimizes the degradation of the drug. The islets were incubated with alloxan for 30min at 4 degrees C and subsequently various aspects of their metabolism were studied. The O(2) consumption was measured by the Cartesian-diver technique. Islets exposed to 2mm-alloxan and control islets had the same endogenous respiration, whereas the O(2) uptake of the alloxan-treated islets was inhibited and that of the control islets stimulated when they were incubated with 28mm-glucose as an exogenous substrate. The islet glucose oxidation was estimated by measurement of the formation of (14)CO(2) from [U-(14)C]glucose at 37 degrees C. Compared with the controls, alloxan-treated islets showed a decrease in the glucose-oxidation rate in a dose-dependent manner. Pretreatment of the islets with 28mm-glucose for 30min at 37 degrees C completely protected against this effect, whereas preincubations at glucose concentrations below 16.7mm failed to exert any protective effect. The glucose utilization was estimated as the formation of (3)H(2)O from [5-(3)H]glucose. Alloxan (2mm) failed to affect islet glucoseutilization rate in the presence of either 2.8 or 28mm-glucose. In contrast, islets exposed to 5 or 10mm-alloxan exhibited lowered glucose utilization. It is concluded that in vitro alloxan has an acute inhibitory effect on the islet glucose metabolism, and that this action can be prevented by previous exposure to a high glucose concentration. The results are consistent with the idea that the B-cytotoxicity of alloxan reflects an interaction with intracellular sites involved in the oxidative metabolism of the B-cell.     

Comparison of Light and Electron Microscopic Determinations of the Number of Bacteria and Algae in Lake Water

Determinations of the number of microorganisms in lake water samples with the bright-field light microscope were performed using conventional counting chambers. Determinations with the fluorescence microscope were carried out after staining the organisms with acridine orange and filtering them onto Nuclepore filters. For transmission electron microscopy, a water sample was concentrated by centrifugation. The pellet was solidifed in agar, fixed, dehydrated, embedded in Epon, and cut into thin sections. The number and area of organism profiles per unit area of the sections were determined. The number of organisms per unit volume of the pellet was then calculated using stereological formulae. The corresponding number in the lake water was obtained from the ratio of volume of solidified pellet/volume of water sample. Control experiments with pure cultures of bacteria and algae showed good agreement between light and electron microscopic counts. This was also true for most lake water samples, but the electron microscopic preparations from some samples contained small vibrio-like bodies and ill-defined structures that made a precise comparison more difficult. Bacteria and small blue-green and green algae could not always be differentiated with the light microscope, but this was easily done by electron microscopy. Our results show that transmission electron microscopy can be used for checking light microscopic counts of microorganisms in lake water.     

Castilleja (Scrophulariaceae) of Costa Rica and Panama

The mountains of Costa Rica and western Panama constitute a natural floristic region that is inhabited by six species ofCastilleja.Castilleja talamancensis, C. lentii andC. tayloriorum are described as new. The remaining three areC. arvensis,C. irasuensis andC. quirosii. All butC. arvensis are endemic to the area. The six species are keyed, described, illustrated, mapped and documented with literature and specimen citations.     

Protein reagent modification of cholera toxin: characterization of effects on antigenic, receptor-binding and toxic properties.

The effects of protein modification procedures on the biologically most important properties of cholera toxin, i.e. the toxic activity, the GM1 receptor-binding capacity and the antigenic (antibody-fixing) properties, have been studied quantitatively using microgram amounts or less of toxin protein. Most of the 24 group-specific reagents used had either no inhibitory effect on the toxic or the combination of GM1-binding and antibody-fixing properties of cholera toxin, or they had a concomitant inhibitory effect on these activities. Separate testing of GM1- and antibody-binding revealed a close, but not absolute, structural association between these properties, Amino group reactive substances were particularly effective in decreasing the GM1-binding activity, while leucine aminopeptidase had no effect. This suggests that lysine residues may be involved in binding toxin to the acidic GM1 receptor. Sodium dodecylsulphate and mercaptoethanol, which caused dissociation of the subunits of cholera toxin as indicated by polyacrylamide gel electrophoresis, abolished toxicity without inhibiting the concomitant GM1- and antibody-binding properties of the toxin. Similar differential effects were also obtained with three reagents which did not seem to change the aggregation state of the toxin. These substances all had specificity for arginine, suggesting that arginyl residues of the toxin molecule may be involved in a 'toxic site' distinct from the receptor-binding site(s). A selective effect on the toxic site was also found by treating the toxin with carboxypeptidase or trypsin in the presence of urea; in the absence of urea no enzymic effect on any toxin property was noted.     

Galanin-like immunoreactivity in extrinsic and intrinsic nerves to the gut of the Atlantic cod,Gadus morhua,and the effect of galanin on the smooth muscle of the gut

The presence of galanin-like immunoreactivity in nerves to the stomach of the Atlantic cod has been investigated by immunohistochemistry. The distribution of ganglion cells showing galanin-like immunoreactivity was compared with the total distribution in nerves and ganglia. Projection studies were made to determine the origin of the galanin neurons. The effect of galanin was studied in smooth muscle strip preparations of the gut wall and arteries. Galanin-like immunoreactive ganglion cells frequently occurred along the vagal branches to the stomach. Most of them projected cranially. Immunoreactive nerve fibres were present in all layers of the gut and around arterial branches on the surface of the stomach. Ligations of the vagus and splanchnic nerves produced accumulations of immunoreactive material on both sides of the ligature. Galanin produced weak contractile effects unaffected by tetrodotoxin on the gut wall and on gut arteries. It is concluded that a population of the ganglion cells along the vagus nerve in the Atlantic cod contains a galanin-like peptide. Some of these cells may be parts of autonomic parasympathetic pathways innervating the gut of the Atlantic cod, having direct excitatory effects on the smooth muscles of the gut wall and gut arteries.     

SOMA: A Single Oligonucleotide Mutagenesis and Cloning Approach

Modern biology research requires simple techniques for efficient and restriction site-independent modification of genetic material. Classical cloning and mutagenesis strategies are limited by their dependency on restriction sites and the use of complementary primer pairs. Here, we describe the Single Oligonucleotide Mutagenesis and Cloning Approach (SOMA) that is independent of restriction sites and only requires a single mutagenic oligonucleotide to modify a plasmid. We demonstrate the broad application spectrum of SOMA with three examples. First, we present a novel plasmid that in a standardized and rapid fashion can be used as a template for SOMA to generate GFP-reporters. We successfully use such a reporter to assess the in vivo knock-down quality of morpholinos in Xenopus laevis embryos. In a second example, we show how to use a SOMA-based protocol for restriction-site independent cloning to generate chimeric proteins by domain swapping between the two human hRMD5a and hRMD5b isoforms. Last, we show that SOMA simplifies the generation of randomized single-site mutagenized gene libraries. As an example we random-mutagenize a single codon affecting the catalytic activity of the yeast Ssy5 endoprotease and identify a spectrum of tolerated and non-tolerated substitutions. Thus, SOMA represents a highly efficient alternative to classical cloning and mutagenesis strategies.