Cloning of DNA involved in sporulation of Streptomyces griseus

Twenty-two bald mutants of Streptomyces griseus were isolated and classified into four phenotypic groups, two of which showed conditional sporulation. A 3-kilobase fragment of DNA was cloned in a high-copy-number vector and detected by its ability to restore sporulation to one class of conditionally bald mutants. Analysis of subclones demonstrated that the sporulation property was contained within a 2.5-kilobase fragment. Hybridization studies and restriction analysis indicated that this DNA fragment was present in several Streptomyces species and was distinct from DNA that has been shown to complement afsA mutants of S. bikiniensis and bldA mutants of S. coelicolor.     

Specific labeling and partial inactivation of cytochrome oxidase by fluorescein mercuric acetate

Addition of 1 eq of fluorescein mercuric acetate (FMA) to beef heart cytochrome oxidase was found to inhibit the steady-state electron transfer activity by 50%, but further additions up to 10 eq had no additional effect on activity. The partial inhibition caused by FMA is thus similar to that observed with other mercury compounds (Mann, A. J., and Auer, H. E. (1980) J. Biol. Chem. 255, 454-458). The fluorescence of FMA was quenched by a factor of 10 upon binding to cytochrome oxidase, consistent with the involvement of a sulfhydryl group. However, addition of mercuric chloride to FMA-cytochrome oxidase resulted in an increase in fluorescence, suggesting that FMA was displaced from the high affinity binding site. Cytochrome c binding to FMA-cytochrome oxidase resulted in a 10% decrease in the fluorescence, possibly caused by Forster energy transfer from FMA to the cytochrome c heme. The binding site for FMA in cytochrome oxidase was investigated by carrying out sodium dodecyl sulfate gel electrophoresis under progressively milder dissociation conditions. When FMA-cytochrome oxidase was dissociated with 3% sodium dodecyl sulfate and 6 M urea, FMA was predominantly bound to subunit II following electrophoresis. However, when the dissociation was carried out at 4 degrees C in the absence of urea with progressively smaller amounts of lithium dodecyl sulfate, the labeling of subunit II decreased and that of subunit I increased. These experiments demonstrate that mercury compounds bind to a high affinity site on cytochrome oxidase, possibly located in subunit I, but then migrate to subunit II under the normal sodium dodecyl sulfate gel electrophoresis conditions. A definitive assignment of the high affinity binding site in the native enzyme cannot be made, however, because it is possible that mercury compounds can migrate from one sulfhydryl to another under even the mildest electrophoresis conditions.     

Spotlight on phytochrome nomenclature

These recommendations for genes encoding phytochromes were developed independently by Quail et al., but are broadly consistent with the Commission's guidelines. Their original article, kindly provided in advance of publication, appeared as a Letter to the Editor inPlant Cell (6:468–471, 1994) and is published with permission of the American Society of Plant Physiologists.     

Relationships among microarthropods,fungi, and their environment

Temporal and spatial relationships in a maple-forest soil among mycophagous microarthropods, total hyphal length, vesicular-arbuscular mycorrhizal (VAM) fungus spores, microfungus diversity, root biomass and some abiotic variables (temperature, water content, pH, organic matter content) were investigated. Samples were obtained from spring 1991 to winter 1992 at four soil depths. Canonical correspondence analysis was used to analyze the data. Four species of sporulating VAM fungi were identified, along with 23 species of mites and springtails, 9 of which were common. Hyphal length, VAM fungus spores, and soil animals peaked in spring and autumn. Canonical correspondence analysis suggests that animal abundance and success in the soil is dependent on a number of environmental variables. The most important variables that influence microarthropod community structure are: (i) temperature, (ii) water content, (iii) pH, (iv) total length of fungal hyphae, and (v) diversity of darkly-pigmented fungi. However, the relative importance of these variables changes with increasing soil depth. We have also shown a relationship between arthropod populations and their food supply under field conditions, a phenomenon that has been demonstrated previously under controlled laboratory conditions.     

Effects of filipin and steroids on phytochrome pelletability

Red light given to dark-grown etiolated leaves of Hordeum vulgare L. in vivo or to crude homogenates increases the phytochrome content of the 20,000 g pellet on centrifugation. The steroids cholesterol and stigmasterol inhibit this red light-induced phytochrome pelletability. Filipin (a polyene antibiotic, which is known to combine with steroids) inhibits red light-induced phytochrome pelletability. Filipin and steroids at the appropriate concentration applied together prevent the inhibition caused by either when applied alone. These results suggest that phytochrome may bind to a steroid component of membranes. The phospholipid phosphatidyl choline dipalmitoyl has no effect on red light-induced phytochrome pelletability. Preliminary evidence demonstrates a direct association of soluble phytochrome in its active form and steroids. The physiological significance of red light-induced pelletability and the primary mechanism of phytochrome action are discussed in terms of a hypothetical steroid-binding site.     

Effect of an exercise run to exhaustion on cAMP in the rat heart

The purpose of this investigation was to determine whether adenosine 3',5'-cyclic monophosphate (cAMP) content is increased in vivo in the heart as a result of exercise at a time when there is rapid cardiac glycogen utilization. Rats were run to exhaustion on a treadmill for a period of 164.5 +/- 9.5 min. Blood norepinephrine and epinephrine were significantly elevated approximately 2.5-fold above resting levels at the end of the treadmill run. Myocardial glycogen was reduced by 54.7% at exhaustion compared with control values. Myocardial cAMP was significantly elevated 88% above control levels as a result of the run. Associated with the depletion of myocardial glycogen and the elevation of cAMP was an activation of phosphorylase to its a form. These data suggest that myocardial glycogen metabolism during exercise is, in part, mediated by hormonal influences that are associated with increases in cAMP.     

Calcium buffering in presynaptic nerve terminals. I. Evidence for involvement of a nonmitochondrial ATP-dependent sequestration mechanism

A latent ATP-dependent Ca storage system is enriched in preparations of pinched-off presynaptic nerve terminals (synaptosomes), and is exposed when the terminals are disrupted by osmotic shock or saponin treatment. The data indicate that a fraction of the Ca uptake (measured with 45Ca) is associated with the intraterminal mitochondria; it is blocked by ruthenium red, by FCCP, and by azide + dinitrophenol + oligomycin. There is, however, a residual ATP-dependent Ca uptake that is insensitive to the aforementioned poisons; this (nonmitochondrial) Ca uptake is blocked by tetracaine, mersalyl and A-23187. Moreover, A-23187 rapidly releases previously accumulated Ca from these (nonmitochondrial) storage sites, whereas the Ca chelator, EGTA, does not. The proteolytic enzyme, trypsin, spares the mitochondria but inactivates the nonmitochondrial Ca uptake mechanism. Chemical measurements of total Ca indicate that the ATP-dependent Ca uptake at the nonmitochondrial sites involves the net transfer of Ca from medium to tissue fragments. This system can sequester Ca when the ambient-ionized Ca2+ concentration (buffered with EGTA) is less than 0.3 micrometer; brain mitochondria take up little Ca when the ionized Ca2+ level is this low. Preliminary subfractionation studies indicate that the nonmitochondrial Ca storage system does not sediment with synaptic vesicles. We propose that this Ca storage system, which has many properties comparable to those of skeletal muscle sarcoplasmic reticulum, may be associated with intraterminal smooth endoplasmic reticulum. This Ca-sequestering organelle may help to buffer intracellular Ca.     

Efficient Lipid Staining in Plant Material with Sudan Red 7B or Fluoral Yellow 088 in Polyethylene Glycol-Glycerol

Polyethylene glycol (400) with 90% glycerol (aqueous) is introduced as an efficient solvent system for lipid stains. Various lipid-soluble dyes were dissolved in this solvent system and tested for their intensity, contrast, and specificity of staining of suberin lamellae in plant tissue. The stability (i.e., lack of precipitation) of the various staining solutions in the presence of fresh tissue was also tested. When dissolved in polyethylene glycol-glycerol, Sudan red 7B (fat red) was the best nonfluorescent stain and fluorol yellow 088 (solvent green 4) was an excellent fluorochrome. These two dyes formed stable staining solutions which efficiently stained lipids in fresh sections without forming precipitates. Estimations of the solubilities of these dyes in the solvent compared with their solubilities in lipids of various chemical types indicated that they should both be effective stains for lipids in general.