Mutational analysis of Escherichia coli σ28 and its target promoters reveals recognition of a composite −10 region, comprised of an 'extended −10' motif and a core −10 element

σ²⁸ controls the expression of flagella-related genes and is the most widely distributed alternative σ factor, present in motile Gram-positive and Gram-negative bacteria. The distinguishing feature of σ²⁸ promoters is a long −10 region (GCCGATAA). Despite the fact that the upstream GC is highly conserved, previous studies have not indicated a functional role for this motif. Here we examine the functional relevance of the GCCG motif and determine which residues in σ²⁸ participate in its recognition. We find that the GCCG motif is a functionally important composite element. The upstream GC constitutes an extended −10 motif and is recognized by R91, a residue in Domain 3 of σ²⁸. The downstream CG is the upstream edge of −10 region of the promoter; two residues in Region 2.4, D81 and R84, participate in its recognition. Consistent with their role in base-specific recognition of the promoter, R91, D81 and D84 are universally conserved in σ²⁸ orthologues. σ²⁸ is the second Group 3 σ shown to use an extended −10 region in promoter recognition, raising the possibility that other Group 3 σs will do so as well.     

Vitamin D3 affects growth and Ca2+ uptake by Phaseolus vulgaris roots cultured in vitro

Vitamin D₃ at low concentration (10⁻⁹ M) inhibited the growth of Phaseolus vulgaris L. (cv. Contrancha) roots in vitro as measured by elongation (14 h) and [³H]-leucine incorporation into protein (2 h), and increased their labelling with ⁴⁵Ca²⁺ (2 h). Cycloheximide and puromycin (50 u.M) blocked vitamin D₃ stimulation of root ⁴⁵Ca²⁺ labelling, indicating that it is mediated by de novo protein synthesis. The calcium ionophore X-537A (10⁻⁵JW) induced similar changes both in root elongation and ⁴⁵Ca²⁺ uptake (14 h). This may indicate that the inhibitory effects of the sterol on root growth are mediated by changes in Ca²⁺ fluxes. However, this interpretation should be further strengthened by additional studies as the ionophore may have acted on root growth, affecting physiological processes other than Ca²⁺ transport.     

Instability of the L6 gene for rust resistance in flax is correlated with the presence of a linked Ac element

In a programme aimed at tagging rust-resistance genes in flax with the maize transposable element Ac , a primary transformant of a line called 'Forge' that is homozygous for four rust-resistance genes, L ⁶, M, N and P ², was identified that possessed 10 copies of the Ac element, one of which was linked (29 map units) to L ⁶. Descendants of this plant, which had from 8 to 15 copies of Ac , were crossed to a rust-susceptible line and the progeny screened for rust-susceptible mutants. When the Ac linked to L ⁶ was present in the parent, a high frequency of L ⁶ mutants was observed (29 mutants in 30 575). By contrast, when this Ac was absent, no such mutants were observed in 9258 progeny. The background frequency of L ⁶ mutants was low (five in 124 088). A detailed analysis was made of the first 11 L ⁶ mutants recovered from parents carrying the L ⁶-linked Ac element. While none of the mutants possessed a tagged resistance gene, all lacked an RFLP marker closely linked to L ⁶, suggesting that deletions were responsible for loss of the L ⁶ specificity. In many of the mutants, one or more RFLP markers in the vicinity of the linked Ac were also absent. These findings suggest that the linked Ac may be inducing chromosome breakage.     

Myelin P0 Glycoprotein: Identification of the Site Phosphorylated In Vitro and In Vivo by Endogenous Protein Kinases

Abstract: Myelin membrane prepared from mouse sciatic nerve possesses both kinase and substrates to incorporate [³²P]PO₄³⁻ from [γ-³²P]ATP into protein constituents. Among these, P₀ glycoprotein is the major phosphorylated species. To identify the phosphorylated sites, P₀ protein was in vitro phosphorylated, purified, and cleaved by CNBr. Two ³²P-phosphopeptides were isolated by HPLC. The exact localization of the sequences around the phosphorylated sites was determined. The comparison with rat P₀ sequence revealed, besides a Lys¹⁷² to Arg substitution, that in the first peptide, two serine residues (Ser¹⁷⁶ and Ser¹⁸¹) were phosphorylated, Ser¹⁷⁶ appearing to be modified subsequently to Ser¹⁸¹. In the second peptide, Ser¹⁹⁷, Ser¹⁹⁹, and Ser²⁰⁴ were phosphorylated. All these serines are clustered in the C-terminal region of P₀ protein. This in vitro study served as the basis for the identification of the in vivo phosphorylation sites of the C terminal region of P₀. We found that, in vivo, Ser¹⁸¹ and Ser¹⁷⁶ are not phosphorylated, whereas Ser¹⁹⁷, Ser¹⁹⁹, Ser²⁰⁴, Ser²⁰⁸, and Ser²¹⁴ are modified to various extents. Our results strongly suggest that the phosphorylation of these serine residues alters the secondary structure of this domain. Such a structural perturbation could play an important role in myelin compaction at the dense line level.     

Trifluoperazine inhibits the incorporation of labelled precursors into lipids, proteins and DNA of Mycobacterium tuberculosis H37Rv

Abstract We have recently demonstrated that the calmodulin antagonist trifluoperazine has antitubercular activity in vitro against Mycobacterium tuberculosis H₃₇R_v susceptible and resistant to isoniazid. It is shown that trifluoperazine at a concentration of 50 μ g ml⁻¹ when added to the cells along with the labelled precursors inhibited the incorporation of [¹⁴C]acetate into lipids (63%) and uptake of [¹⁴C]glycine (74%) and [^3H]thymidine (52%) bu whole cells of M. tuberculosis H₃₇R_v by 6 h of exposure. After 48 h, the inhibition was 87%, 97% and 74%, respectively. However, when the drug was added to cells taking up and metabolizing the labelled precursors at a later point (3 h for [¹⁴C]acetate and [³H]thymidine and 12 h for [¹⁴C]glycine) it inhibited completely the uptake of all the precursors, at least up to 24 h. The onset of inhibitory action was very rapid, i.e. 3 h. It is suggested that trifluoperazine has multiple sites of action and acts probably by affecting the synthesis of lipids, proteins and DNA.     

Methane emission from Texas rice paddy soils. 2. Seasonal contribution of rice biomass production to CH4 emission

Measurements focused on seasonal contribution of rice productivity to methane emission were made in three experiments conducted in Texas flooded paddy soils during 1994 and 1995 growing seasons. A total of five rice cultivars representing two distinct groups in methane emission were involved. Over a 10-week period after permanent flooding, total seasonal methane emission was positively correlated with rice above-ground biomass ( r ² = 0.845, n = 11). A very strong dependence of daily methane emission on above-ground vegetative biomass ( r ² = 0.887, n = 93) and on root biomass ( r ² = 0.816, n = 33) was also observed. Calculation from three developmental periods (vegetative, reproductive and ripening) of rice plant indicated that more than 75% of total seasonal methane was emitted during the last 5-week period in concert with reproductive and ripening stages, while rice biomass production during the same period amounted to ≈ 50% of the seasonal total. According to the correlation of cumulative methane emission with above-ground biomass increment between every two-week interval ( r ² = 0.490, n = 93, P = 0.000), the carbon released as methane is approximately equivalent to 3% and 4.5% of photosynthetically fixed carbon in the biomass for low and high emission cultivars, respectively. A further investigation showed that these fractions are related to plant growth and development. The carbon ratio of methane emitted to net photosynthetic production during vegetative, reproductive, and ripening periods averaged 0.9%, 3.6% and 7.9%, respectively, for low emission cultivars, and 2.0%, 5.0% and 8.3%, respectively, for high emission cultivars. Moreover, the ratio was strongly dependent on plant biomass, resulting in r 2 values from 0.775 to 0.907.     

Adaptation of plasma membrane H+-ATPase of rice roots to low pH as related to ammonium nutrition

The preference of paddy rice for NH₄⁺ rather than NO₃^- is associated with its tolerance to low pH since a rhizosphere acidification occurs during NH₄⁺ absorption. However, the adaptation of rice root to low pH has not been fully elucidated. This study investigated the acclimation of plasma membrane H⁺-ATPase of rice root to low pH. Rice seedlings were grown either with NH₄⁺ or NO₃^-. For both nitrogen forms, the pH value of nutrient solutions was gradually adjusted to pH 6.5 or 3.0. After 4 d cultivation, hydrolytic H⁺-ATPase activity, V _max, K _m, H⁺-pumping activity, H⁺ permeability and pH gradient across the plasma membrane were significantly higher in rice roots grown at pH 3.0 than at 6.5, irrespective of the nitrogen forms supplied. The higher activity of plasma membrane H⁺-ATPase of adapted rice roots was attributed to the increase in expression of OSA1, OSA3, OSA7, OSA8 and OSA9 genes, which resulted in an increase of H⁺-ATPase protein concentration. In conclusion, a high regulation of various plasma membrane H⁺-ATPase genes is responsible for the adaptation of rice roots to low pH. This mechanism may be partly responsible for the preference of rice plants to NH₄⁺ nutrition.     

Dominant ptsH mutations of the gene coding for synthesis of the HPr protein of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli K-12 merodiploids

Abstract The Escherichia coli ptsI and ptsH genes code for the synthesis of two proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), namely enzyme I and protein HPr. A number of ptsI ⁺ ptsH ⁺/F' ptsI ⁺ ptsH merodiploids was obtained. It was shown in experiments in vivo that ptsH mutations in the transposition are dominant. Bacterial extracts from these merodiploids supported [¹⁴C]methyl glucoside (MG) phosphorylation at the expense of phosphoenolpyruvate only half as much as extracts from the pts ⁺ cells. ptsI ⁺ ptsH /F' ptsI ⁺ ptsH ⁺ merodiploids appeared to be non-viable; the reason for this lack of viability is discussed.     

Ribonucieic Acid and Protein Metabolism of t12/t12 Embryos and T/t12 Spermatozoa

RNA precursor uptake and incorporation, amino acid uptake and incorporation, and the characterization of newly synthesized RNA and protein in pools of normal morulae and pools containing one-third t ¹²/ t ¹² morulae were compared. Maturing spermatoza of +/+ and T / t ¹² animals were analyzed for RNA and protein content, and the RNA characterized. No differences in these parameters could be ascribed to the t ¹² gene in homozygous embryos or haploid sperm.     

Cadmium-induced changes in the composition and structure of the light-harvesting chlorophyll a/b protein complex II in radish cotyledons

Five-day-old etiolated radish ( Raphanux salivux L. cv. Saxa) seedlings exposed to white continuous light in the presence of Cd²⁺ (0.2 mM) showed characteristic changes in their light-harvesting chlorophyll a/b protein complex II after 48 h of greening. The content of its oligomeric supramolecular form was greatly diminished with a concomitant increase in the level of the monomer. The isolation of highly purified light-harvesting chlorophyll a/b protein complex II from control and Cd²⁺ treated radish cotyledons and a detailed analysis of its structure and composition revealed that first of all, Cd²⁺ altered the content of the specific phosphatidylglycerol fatty acid - trans -Δ³-hexadecenoic acid, widely accepted as a component responsible for the oligomerization of this chlorophyll-protein complex. This fatty acid in the thylakoid membrane phosphatidylglycerol pool seems to be very sensitive to different environmental stresses lowering its content, which indicates the vital significance of this component for the supramolecular organization and proper functioning of the light-harvesting chlorophyll a/b protein complex II.     

Characterization of amplified esterase Estβ12 associated with organophosphate resistance in a multi-resistant population of the mosquitoCulex quinquefasciatus from Cuba

Esterase amplification is the major organophosphorus (OP) insecticide resistance mechanism in Culex mosquitoes. The amplified Estα2 ^1\ Estβ2 ¹ esterases are found in > 90% of resistant populations worldwide, whereas amplified DNAs (amplicons) containing Estβ1s are much rarer. Individuals with the Estβ1 amplicons appear to be at a selective disadvantage in competition with those carrying the Estα2 ¹\ Estβ2 ¹ amplicons. To test the hypothesis that this is because Estβ1 is less able to bind insecticide than the common amplified esterases, Estβ1² was purified from the multi-resistant Habana strain of Culex quinquefasciatus , from Cuba. In its native form Estβ1 is a monomeric enzyme of 66 kDa, with a pI of 4.8. The bimolecular rate constants for interaction of Estβ1² with several OP insecticides were similar to those for the commonly elevated esterases Estα2¹ and Estβ2¹, and much higher than for the electrophoretically identical non-elevated Estβ1³ and Estα3. Hence the apparent selective advantage of the Estα2 ¹\ Estβ2 ¹ amplicon is not due to its greater efficiency of insecticide binding, as OP insecticides are significantly better inhibitors of all the amplified esterases than of their non-amplified counterparts and therefore should be equally effective at conferring resistance.     

Effects of Monensin on Assembly of Po Protein into Peripheral Nerve Myelin

Abstract: The ionophore monensin has been used in a variety of systems to block secretion of glycoproteins or assembly of glycoproteins into membranes. We examined the effects of monensin on assembly of the Po glycoprotein into PNS myelin, and compared this agent with the glycosylation inhibitor tunicamycin in our system. Sciatic nerves from 9-day-old rat pups were sliced and incubated in vitro . Electron microscopy of the Schwann cells in slices incubated with monensin revealed extensive swelling of the Golgi complex. Incubation with 10⁻⁷ M monensin inhibited total protein synthesis by about 20% and fucose incorporation into protein about 35%. Following isolation of myelin, proteins were separated by sodium dodecyl sulfate gel electrophoresis. Monensin inhibited the appearance of Po in myelin, while causing its accumulation in a denser membrane fraction. In addition, a slightly faster-migrating species of P_o labeled with both [³H]fucose and [¹⁴C]glycine was observed in all fractions. Assembly of basic proteins into myelin was not affected. Preincubation with 10 μg/ml tunicamycin for 30 min prior to incubation with [³H]fucose and [¹⁴C]glycine for 2 h resulted in a 65% decrease in [³H]fucose incorporation into P_o, and the appearance of a new [¹⁴C]glycine-labeled peak that migrated in the region of the 23K protein reported by Smith and Sternberger. [³H]Fucose incorporation was inhibited earlier, and to a greater extent, than protein synthesis. Our results show that processing of the P_o glycoprotein is sensitive to both monensin and tunicamycin, and that monensin partially blocks assembly of P_o into myelin.     

Kinetics of Entry of P0 Protein into Peripheral Nerve Myelin

Abstract: Sciatic nerves from 9-day-old rat pups were removed, sliced into 0.4-mm sections, and incubated with [³H]fucose or [¹⁴C]glycine precursors. The nerve slice system gave nearly linear incorporation of [³H]fucose as a function of time for 3 h, after an initial lag of ˜30 min for homogenate and ˜60 min for myelin. Incorporation of [³H]fucose at constant specific radioactivity was directly proportional to exogenous fucose levels over the range 3.0 × 10⁻⁸ m to 1.5 × 10⁻⁶ m . Analysis of labeled proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that greater than 50% of labeled glycoprotein was P₀, with no other major constituents. This system was used in fucose-chase experiments to determine that a period of ˜20 min elapses between fucosylation and assembly of P₀ into myelin. Cycloheximide inhibition of protein synthesis was used to determine that a period of ˜33 min elapses between protein synthesis and appearance of P₀ myelin.     

Melatonin Effect on [3H]Glutamate Uptake and Release in the Golden Hamster Retina

Abstract: The effect of melatonin on [³H]glutamate uptake and release in the golden hamster retina was studied. In retinas excised in the middle of the dark phase, i.e., at 2400 h, melatonin (0.1 and 10 n M ) significantly increased [³H]glutamate uptake, and this effect persisted in a Ca²⁺-free medium. On the other hand, melatonin significantly increased [³H]glutamate release in retinas excised at 2400 h, but this effect was Ca²⁺ sensitive. Melatonin significantly increased ⁴⁵Ca²⁺ uptake by a crude synaptosomal fraction from retinas of hamsters killed at 2400 h. In retinas excised at 1200 h, melatonin had no effect on [³H]glutamate uptake, [³H]glutamate release, or ⁴⁵Ca²⁺ uptake at any concentration tested. Cyclic GMP analogues, i.e., 8-bromoguanosine 3',5'-cyclic monophosphate and 2'- O -dibutyrylguanosine 3',5'-cyclic monophosphate, significantly increased [³H]glutamate uptake, [³H]glutamate release, and ⁴⁵Ca²⁺ uptake by tissue removed at 1200 and 2400 h, suggesting that the effects of melatonin could correlate with a previously described effect of melatonin on cyclic GMP levels in the golden hamster retina. Taking into account the key role of glutamate in visual mechanisms, the results suggest the participation of melatonin in retinal physiology.     

Cloning, molecular characterization and heterologous expression of AMY1, an α-amylase gene from Cryptococcus flavus

A Cryptococcus flavus gene ( AMY1 ) encoding an extracellular α-amylase has been cloned. The nucleotide sequence of the cDNA revealed an ORF of 1896 bp encoding for a 631 amino acid polypeptide with high sequence identity with a homologous protein isolated from Cryptococcus sp. S-2. The presence of four conserved signature regions, (I) ¹⁴⁴DVVVNH¹⁴⁹, (II) ²³⁵GLRIDSLQQ²⁴³, (III) ²⁶³GEVFN²⁶⁷, (IV) ³²⁷FLENQD³³², placed the enzyme in the GH13 α-amylase family. Furthermore, sequence comparison suggests that the C. flavus α-amylase has a C-terminal starch-binding domain characteristic of the CBM20 family. AMY1 was successfully expressed in Saccharomyces cerevisiae . The time course of amylase secretion in S. cerevisiae resulted in a maximal extracellular amylolytic activity (3.93 U mL⁻¹) at 60 h of incubation. The recombinant protein had an apparent molecular mass similar to the native enzyme ( c . 67 kDa), part of which was due to N-glycosylation.