Cloning,expression, and nucleotide sequence of livR,the repressor for high-affinity branched-chain amino acid transport in Escherichia coli

The livR gene encoding the repressor for high-affinity branched-chain amino acid transport in Escherichia coli has been cloned from a library prepared from the episome F106. The inserted DNA fragment from the initial cloned plasmid, pANT1, complemented two independent, spontaneously derived, regulatory mutations. Subcloning as well as the creation of deletions with Bal31 exonuclease revealed that the entire regulatory region is contained within a 1.1-kb RsaI-SalI fragment. Expression of the pANT plasmids in E. coli minicells showed that the regulatory region encodes one detectable protein with an apparent molecular weight of 21,000. DNA sequencing revealed one open reading frame of 501 bp encoding a protein with a calculated MW of 19,155. The potential secondary structure of the regulatory protein has been predicted and it suggests that the carboxy terminus may fold into three consecutive alpha helices. These results suggests that the livR gene encodes a repressor which plays a role in the regulation of expression of the livJ and the livK transport genes.     

Partial purification and characterization of two soluble c-type cytochromes from Chromatium vinosum

Two c-type cytochromes from Chromatium vinosum have been partially purified and characterized. Cytochrome c₅₅₀, which appears to function as an electron carrier in the cyclic electron transport chain of this photosynthetic purple sulfur bacterium, has a molecular weight of approximately 15,000 and an oxidation-reduction midpoint potential (E_m) of + 240 mV at pH 7.4. It has (in the reduced form) an α band at 550 nm and a β band at 520 nm. Cytochrome c₅₅₁ is characterized by absorbance maxima at 354 and 409 nm in the oxidized form and 418, 523, and 551 nm in the reduced form. The reduced cytochrome reacts with CO. Cytochrome c₅₅₁ is a monomeric protein with a molecular weight of 18,800 ± 700 and E_m = ?299 ± 5 mV (pH independent between pH 6.3 and 8.0). It appears to lack a methionine axial ligand as indicated by the absence of an absorbance band at 695 nm in the oxidized form.     

Purification and Characterization of Myosin from Calf Brain

Actomyosin complex was extracted from the brain cortex in a medium consisting of low salt, ATP, and EDTA, in the presence of protease inhibitors, followed by ammonium sulfate fractionation. Myosin was then purified from the actomyosin. Myosin obtained according to the procedure used was significantly contaminated with actin high (greater than 200,000 dalton) and low molecular weight proteins. Therefore, an alternative method based on affinity chromatography (Blue Dextran/Sepharose) and gel filtration (Sepharose 4B) was developed to purify myosin. This procedure yielded myosin that was greater than 95% pure as judged by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit composition of purified brain myosin was monitored by sodium dodecyl sulfate-polyacrylamide gel also containing a urea gradient. A closely migrating triplet in the heavy chain and three light chains, LC1, LC2, and LC3, of Mr 21,000, 19,000, and 17,000, respectively, were observed. These findings raise the possibility of the existence of myosin isoenzymes in the brain. Brain myosin formed bipolar thick filaments in 0.075 M KCl and MgCl2. At low ionic strength, the Mg2+-ATPase activity of myosin was stimulated 3- to 3.5-fold in the presence of skeletal muscle f-actin. Brain myosin also hydrolyzed other nucleotides; the rate of hydrolysis was ITP greater than ATP approximately equal to CTP greater than GTP approximately equal to UTP. The substrate (ATP) saturation curve in the presence of 10 mM CaCl2 and 0.6 M KCl was complex and consisted of plateau regions. The Arrhenius plot of the Ca-ATPase data was linear, whereas with ITPase, it was biphasic with a break occurring around 20 degrees C.     

Bicarbonate transport inChara corallina: evidence for cotransport of HCO 3 − with H+

Summary Experiments were undertaken on the fresh water algaChara corallina to determine the form of inorganic carbon (CO₂ or HCO ₃ ^– ) which enters the cell during photosynthesis at alkaline pH. Recent proposals have centered on the possibility that proton efflux in alkaline solution is able to generate, in the immediate vicinity of the cell, a sufficiently low pH to raise the partial pressure of CO₂, and hence facilitate its passive permeation into the cell. Predictions have been made by modelling this situation (N.A. Walker, F.A. Smith & I.R. Cathers, 1980,J. Membrane Biol. 5751–58, J.M. Ferrier, 1980,Plant Physiol. 661198–1199), and these were tested by placing recessed-tip pH microelectrodes in the unstirred layer surrounding cells in stagnant solution (bulk pH 8.2, buffered only with 1mm HCO ₃ ^– ). Even as close as 2 m from the cell wall, the pH was typically 7.2 to 7.6 in the acid band center — over 1 pH unitgreater than that suggested by the models for CO₂ entry at the necessary rate for C-fixation. Further evidence for the entry of HCO ₃ ^– , rather than CO₂, at high solution pH was obtained from experiments in which the radial pH gradient in the unstirred layer was reduced. Buffer solutions containing 5mm phosphate or 5mm HEPES, raised the pH at the cell surface in the acid regions from around 7.2 to 7.8 or higher. This pH increase (reduction in acid gradient) would have greatly reduced the CO₂ level at the cell surface and should, therefore, have greatly reduced the CO₂-related¹⁴C-influx. However,¹⁴C-fixation was reduced by only 31% (phosphate) or 15% (HEPES), compared with buffer-free controls. Reduction of the unstirred layer thickness by fast solution flow resulted in a stimulation, and not a reduction, of¹⁴C-fixation. The similarity of our radial pH profiles near the wall with that predicted by the model (Walker et al., 1980) assuming H⁺–HCO ₃ ^– cotransport, together with the effects of buffer, and the results of increased solution flow rate, lead to the conclusion that cotransport of HCO ₃ ^– with H⁺ is the likely method of entry of inorganic carbon. Longitudinal pH profiles of theChara cell were obtained at a distance of 25 m from the wall. These revealed much sharper delineation of the acid and alkaline bands than has previously been possible with miniature pH electrodes. Profiles of local electric field, obtained with a vibrating probe, were in excellent agreement with the high resolution pH profiles. This supports the hypothesis that membrane proton transport has a role (direct) in the generation of the extracellular currents.     

Quantitation of in situ hybridization of ribosomal ribonucleic acids to human diploid cells

The hybridization of 5S and 28S ribosomal RNAs to human fibroblast and leukocyte cells was used as a model system to quantitate the technique of in situ hybridization for human diploid cell types. Quantitation consisted of counting (scoring) the number of grains formed over both interphase nuclei and metaphase chromosomes on slides after various hybridization procedures. The average number of grains/nucleus per slide was then used to determine hybridization percentages. As with nitrocellulose filter hybridizations the kinetics of in situ hybridizations can be fit with a single first-order rate constant. However, the in situ hybridization rate was approximately 10 times slower than the corresponding filter hybridization rate. The efficiency of in situ hybridization was found to range between 5 and 15% for both leukocyte and fibroblast cell types and for both metaphase and interphase nuclei. Determination of the parameters of the in situ hybridization reaction of ribosomal RNAs to diploid chromosomes define the experimental conditions needed for the localization of single copy genes to diploid chromosomes.     

Effects of pH,phosphate and ammonia on the rate of uptake of nitrate and ammonia by freshwater phytoplankton

The effect of phosphate (PO₄ ⁺³) and pH in regulating nitrate (NO₃) and ammonia (NH₃ ⁺) uptake by phytoplankton was investigated in two Oklahoma lakes using ¹⁵N tracers. Addition of PO₄ ⁺³ above ambient concentrations had a negligible effect on the rate of uptake of NO₃ ^– or NH₃ ⁺. Manipulation of pH of lake water had little effect on uptake of either NO₃ ^– or NH₃ ⁺. A correlation analysis suggested that NO₃ ^– is not used by phytoplankton when NH₃ ⁺ concentrations exceed about 210 µg NH₃ ⁺-N(1)^–1.     

Hydrostatic pressure as the controlling factor in the depth distribution of Eurasian watermilfoil,Myriophyllum spicatum L.

These experimental studies have shown that this plant will grow successfully at pressures encountered in water at depths as great as 17 m. When there were suitable levels of light, temperature, nutrients and aeration, the plants grown under constant hydrostatic pressure for three weeks showed variations in the measured amounts of new growth but no measure could be associated with the constant increased hydrostatic pressure. Sudden changes in pressure are thought to play a significant role in aquatic plant growth under experimental conditions.     

A null mutation at the mouse Phosphoglucomutase-1 locus and a new locus,Pgm-3

A null mutation at the phosphoglucomutase locus (Pgm-1) was discovered by electrophoretic analysis of the inbred mouse strain C57 BL/6J. The null allele (Pgm-1 ⁿ) was shown to segregate as a Mendelian unit alternative to the Pgm-1 ^a and Pgm-1 ^b alleles. Mice expressing the Pgm-1 ⁿ allele, either in the heterozygous or homozygous state, are viable, healthy, and fertile. The occurrence of the Pgm-1 ⁿ mutant revealed a previously unreported genetic locus (Pgm-3) that controls the expression of a third phosphoglucomutase. Two electrophoretically expressed alleles of Pgm-3 (inherited without dominance) are found in the inbred mouse strains C57 BL/6J and DBA/2J. Linkage observed between the Pgm-3 locus, the dilute locus (d) and the cytoplasmic malic enzyme locus (Mod-1) has allowed assignment of the Pgm-3 locus to chromosome 9. A striking tissue specific expression of Pgm-1 and Pgm-3 was observed. Products of the Pgm-3 locus were detected in kidney, testes, brain, and heart. In contrast, Pgm-1 controlled isozymes were present in kidney, spleen, ovaries, and erythrocytes.Financial support for this work was provided in part by Contract #263-78-C-0393 from the National Institute of Environmental Health Sciences to the Research Triangle Institute.     

The apparent viscosity of the protoplasm of subepidermal stem basis cells of Pisum sativum. Relation to aging, drought tolerance and water stress

Plants of Pisum sativum L. cv. Alaska wilt resistant were subjected to two different water stress regimes under controlled environment conditions: watering was stopped either on the 7th day (early stress) or on the 21st day (late stress) after planting. Plants under the early stress regime developed drought tolerance (adapted), while those under late stress did not. The apparent viscosity of the protoplasm of subepidermal stem basis cells was analyzed by the centrifugation and plasmolysis form method during the entire growth period.
The apparent viscosity of the subepidermal stem basis cells changed with plant age and was highest in 3-week-old plants. In controls the relation of apparent viscosity to age was the same when measured under full turgor and in relaxed state. Under early stress condition, however, the pattern of the viscosity changes with plant age was significantly different for turgescent and relaxed cells. In four week old plants, a higher apparent viscosity was measured in relaxed adapted cells than in relaxed control cells. It is suggested that the higher apparent viscosity is the result of a delayed cell aging.
Apparent viscosity was inversely proportional to soil moisture content and the osmotic potential of the cell sap for the cells of late stress plants, whereas no clear relation was found for the cells of early stress plants. This difference may indicate two mechanisms of viscosity changes: 1) osmotic dehydration of the protoplasm under water stress (passive viscosity change), 2) changes in the amount, hydration or architecture of macromolecules present in the cytoplasm (active viscosity change). Whereas differences in the apparent viscosity between control and stressed cells may not be the cause of drought tolerance, they seem to indicate the development of drought tolerance. Water stress history and plant age were the most critical factors controlling the apparent viscosity changes observed.     

Maturation and germination of Phaseolus vulgaris embryonic axes in culture

In this study of embryo development in Phaseolus vulgaris L., we found that immature embryonic axes placed in culture show a growth lag before germinating. The length of this lag phase varies according to axis age at excision, but is not affected by transfer to fresh medium, alteration of sucrose concentration between 0.5 and 2%, or whether the culture medium is liquid or agar-solidified. The lag phase was shortened by both actinomycin D and cordycepin treatment, and by treatment with 10^-5 to 10^-6 M benzyladenine. The effect of abscisic acid (ABA) varied with concentration: below a certain level, it had no effect on the lag phase, but above that level it inhibited, germination. This threshold concentration was 10^-7 M for 20-d-old axes but increased to 10^-5 M by the time the axes were 32 to 34 d old. To determine whether the axes were continuing their embryonic development during the lag phase, we tested them for desiccation-tolerance and for synthesis of phaseolin, a seed storage protein which is specific for embryos of P. vulgaris. The ability to germinate after rapid desiccation was acquired by axes at 26 d past anthesis; when axes younger than this were placed in culture, they developed desiccation-tolerance during the lag phase of growth, indicating that they were continuing embryonic maturation. Phaseolin was present in isolated axes, although at lower levels than in cotyledons. It accumulated during axis development in parallel with total protein, staying at about 1% of total protein content. When isolated immature axes were pulsed with ³H-or ¹⁴C-amino acids, they incorporated label into phaseolin, shown by precipitation with anti-phaseolin antibody. Isolated axes from mature seeds, however, did not synthesize detectable amounts of phaseolin. Immature axes cultured in vitro for a period of one to several days continued synthesizing phaseolin until the day prior to visible germination. Treatment of cultured axes with ABA increased the amount of precursor amino acids incorporated into protein, but had a small or no effect on the relative proportion of phaseolin synthesized. We conclude that P. vulgaris axes in culture continue to develop embryonically for a period of time which seems to be under intrinisc control by the axis. This contrasts with precocious germanation, a pattern of embryo behavior seen in many other species. When such embryos are excised from seeds while immature and placed in culture, they switch promptly from embryo development into germination. If ABA or water stress is responsible for preventing precocious germination, it may be that a high level of ABA is maintained or synthesized internally by embryonic axes of Phaseolus, while in other embryos the maternal environment supplies ABA and/or causes water stress.Abbreviations ABA Abscisic acid - BA benzyladenine