Transformation of Methanococcus maripaludis and identification of a Pst I-like restriction system

Abstract An optimized polyethylene glycol (PEG) method of transformation was developed for Methanococcus maripaludis using the pKAS102 integration vector. The frequency of transformation with 0.8 μg of plasmid and 3×10⁹ cells was 4.8×10⁻⁵ transformants cfu⁻¹, or 1.8×10⁵ transformants μg⁻¹, which was four orders of magnitude greater than with the natural transformation method. A Pst I restriction activity in M. maripaludis was also identified. Methylation of the plasmid with Pst I methylase increased the methanococcal transformation frequency at least four-fold. Also, chromosomal DNA from M. maripaludis was resistant to digestion by the Pst I endonuclease.     

Stimulation of shoot elongation in Salix pentandra by gibberellin A9; activity appears to be dependent upon hydroxylation to GAl via GA20

Cessation of shoot elongation in seedlings of Salix pentandra L. is induced by short photoperiod. Gibbereliin A₉ (GA₉) applied either to the apical bud or injected into a mature leaf, induced shoot elongation under a short photoperiod of 12 h, and GA₉ could completely substitute for a transfer to a long photoperiod. When [³H]GA₉ or [²H₂]GA₉ was injected into a leaf, no [³H]GA₉ was detected in the elongating apex and only traces of [³H]GA₉ were found in the shoot above the treated leaf. By the use of gas chromatography-mass spectrometry (GC-MS), [²H₂]GA₂₀ was identified as the main metabolite of [²H₂]GA₉ in both the shoot and the treated leaf. In addition, [²H₂]GA₁ and [²H₂]GA₂₉ were also identified as metabolites of [²H₂]GA₉. These results are consistent with the hypothesis that exogenous GA, promotes shoot elongation in Salix through its metabolism to GA₂₀ and GA,.     

Carbon dioxide assimilation during postharvest removal of astringency from persimmon fruit

Application of anaerobic conditions with CO₂ or N₂ atmospheres to remove astringency from harvested persimmon fruit ( Diospryros kaki L. cv. Triumph), caused production of more acetaldehyde under CO₂ than under N₂, ¹⁴CO₂ applied in a 100% CO₂ atmosphere, for 48 h to astringent persimmon fruits was incorporated mainly into malate and very little into other metabolites, such as carbohydrate or amino acids. Application of malate or pyruvate to pulp discs of astringent persimmons caused an immediate rise in acetaldehyde production. The higher levels of acetaldehyde produced by whole fruits held in a CO₂ atmosphere, than by fruits held in a N₂ atmosphere, can be explained through fixation of atmospheric CO₂ into malate, leading to acetaldehyde production.     

Detection of hydrogen peroxide produced by meat lactic starter cultures

Twelve strains of meat lactic starter cultures (Pediococcus spp. and Lactobacillus plantarum) were found to produce hydrogen peroxide in vitro. The (cumulative) amounts of H₂O₂ produced were measured through the peroxidative action of catalase on H₂O₂ and oxidation of added formate to CO₂ by the H₂O₂-catalase complex formed. There was a problem in building a calibration curve for converting values of formate oxidation into amounts of H₂O₂, either by adding H₂O₂ directly to the assay mixture or having it produced via a glucose-glucose oxidase system.     

Nitrite in the root zone and its effects on ion uptake and growth of wheat seedlings

The immediate and posteffects of various concentrations of NaNO₂ on ion uptake of wheat ( Triticum aestivum L. cv. GK Öthalom) seedlings were studied at different pH values. Without pretreatment, the higher the concentration of NaNO₂ the greater was the decrease in uptake of K⁺ into the roots, both at pH 4 and pH 6. At pH 6 but not at pH 4 the reverse was true when the seedlings were pretreated with NaNO₂. Due to the high Na⁺ content of the roots, an effect of Na⁺ in this process cannot be excluded. Nitrite was taken up by the roots more rapidly than nitrate. Nitrite at 0.1 m M in the medium induced the development of an uptake system for both NO⁻₂ and NO⁻₃ in wheat roots. At higher concentrations pretreatment with NO⁻₂ decreased NO⁻₃ uptake by the roots, but NO₃ did not inhibit the uptake of NO₂. The toxic effect of NO⁻₂ was strongly pH dependent. Lower pH of the external solution led to an increased inhibition by NO⁻₂ of both ion uptake and growth of seedlings. The inhibitory effect of NO⁻₂ differed considerably for roots and shoots. The roots and especially the root hairs were particularly sensitive to NO⁻₂ treatment.     

EFFECTS OF ACTINOMYCIN D AND PUROMYCIN ON THE CELL PROGRESS FROM M TO G1 AND S STAGES IN CULTURED MOUSE LEUKEMIA L5178Y CELLS

Actinomycin D (0.5 μg/ml) did not prevent M stage cells from entering G₁ stage, but blocked their progress from G₁ to S stage. The position of the block was approximately 1.4 hr before S stage or just after the beginning of G₁ stage. Actinomycin D in this concentration also significantly depressed uridine-³H uptake into G₁ stage cells, but did not suppress leucine-³H uptake by M and G₁ cells. This suggests that some proteins may be synthesized in M and G₁ stage cells by messenger RNA left over from the previous cell cycle. However, entry of G₁ cells into S stage would require synthesis of new messenger RNA near the beginning of G₁ stage. Puromycin (10 μg/ml) did not prevent M cells from entering G₁ stage, but blocked their progress from G₁ to S stage. The site of blockage was about 0.7 hr before S stage or in the first two-third of G₁ stage. This might be the site where the cells synthesize new G₁ proteins necessary for entry to S stage.
Comparison of sensitivities of G₁ and G₂ stages to the two antibiotics reveals that the puromycin sensitivity of G₁ cells was similar to that of G₂ cells, but the actinomycin D sensitivity of G₁ was greater than that of G₂ cells.     

Effects of different gibberellins on elongation growth under short day conditions in seedlings of Salix pentandra

Fifteen different gibberellins (GA's) were tested for their ability to induce elongation growth under short day conditions in seedlings of Salix pentandra L. GA's were applied either to the apex or they were injected into a mature leaf. GA₃ was highly active and also GA₄₊₇ and GA₄ showed high activity. GA₁, GA₂, GA₅, GA₉, GA₁₃, GA₂₀, GA₃₆ and GA₄₇ showed moderate activity. GA₁₆, GA₁₇, GA₂₇ and GA₄₁ exhibited low or no activity in doses up to 10 μg per plant. In general, a better growth response was obtained with an application to the apex than with an injection into the leaf.     

B1 and B2 Bradykinin Receptors Encoded by Distinct mRNAs

Abstract: Bradykinin receptors have been subdivided into at least two major pharmacological subtypes, B₁ and B₂. The cDNAs encoding functional B₂ receptors have recently been cloned, but no molecular information exists at present on the B₁ receptor. In this article, we describe experiments examining the possible relationship between the mRNAs encoding the B₁ and B₂ types of receptor. We showed previously that the Human fibroblast cell line W138 expresses both B₁ and B₂ receptors. In this report, we describe oocyte expression experiments showing that the B₁ receptor in W138 human fibroblast cells is encoded by a distinct mRNA ∼2 kb shorter than that encoding the B₂ receptor. We have used an antisense approach in conjunction with the oocyte expression system to demonstrate that the two messages differ in sequence at several locations throughout the length of the B₂ sequence. Taken together with the mixed pharmacology exhibited in some expression systems by the cloned mouse receptor, the data indicate that B₁-type pharmacology may arise from two independent molecular mechanisms.     

Oxygen and the regulation of nitrogen fixation in legume nodules

In N₂-fixing legume nodules, O₂ is required in large amounts for aerobic respiration, yet nitrogenase, the bacterial enzyme that fixes N₂, is O₂ labile. A high rate of O₂ consumptition and a cortical barrier to gas diffusion work together to maintain a low, non-inhibitory O₂ concentration in the central, infected zone of the nodule. At this low O₂ concentration, cytosolic leghemoglobin is required to facilitate the diffusion of O₂ through the infected cell to the bacteria. The resistance of the cortical diffusion barrier is variable and is used by legume nodules to regulate the O₂ concentration in the infected cells such that it limits aerobic respiration and N₂ fixation at all times. The resistance of the diffusion barrier and therefore the degree of O₂ limitation seems to be regulated in response to changes in the O₂ concentration of the central infected zone, the supply of phloem sap to the nodule, and the rate of N assimilation into the end products of fixation.     

Competition between reductive acetogenesis and methanogenesis in the pig large-intestinal flora

Washed bacterial suspensions obtained from the pig hindgut were incubated under ¹³CO₂ in a buffer containing NaH¹³CO₃ and carbohydrates. Incorporation of ¹³C into short chain fatty acids was assayed by quantitative nuclear magnetic resonance. The effects of different levels of H₂ added to the gas phase (0, 20 and 80% v/v) and of the specific methanogenesis inhibitor 2-bromoethane-sulphonic acid (BES) were determined. In control incubations increasing the concentration of H₂ markedly increased methane production. Single- and double-labelled acetate and butyrate were formed in all incubations. In the absence of BES, increasing H₂ significantly increased the incorporation of ¹³CO² into butyrate and the proportion of double-labelled acetate in total labelled acetate. The addition of BES proved to be very successful as a methane inhibitor and greatly enhanced the amount of mono- and double-labelled acetate, especially at the highest H₂ partial pressure. The results suggest that methanogenesis inhibited both routes of reductive acetogenesis, i.e. the homoacetate fermentation of hexose (represented for the most part by single labelling) and the synthesis of acetate from external CO₂ and H₂ (represented mostly by double labelling). A highly significant interaction between BES and H₂ concentration was observed. At the highest pH2 BES increased the proportion of labelled acetate in total acetate from 17.1% for the control to 50.9%. It was concluded that although acetogenesis and methanogenesis can occur simultaneously in the pig hindgut, reductive acetogenesis may become a significant pathway of acetate formation in the absence of methanogenesis.     

Carbon dioxide-concentrating mechanism and the development of extracellular carbonic anhydrase in the marine picoeukaryote Micromonas pusilla

A range of marine photosynthetic picoeukaryote phytoplankton species grown in culture were screened for the presence of extracellular carbonic anhydrase (CA_ext), a key enzyme in inorganic carbon acquisition under carbon- limiting conditions in some larger marine phytoplankton species. Of the species tested, extracellular carbonic anhydrase was detected only in Micromonas pusilla Butcher. The rapid, light-dependent development of CA_ext when cells were transferred from carbon-replete to carbon-limiting conditions was regulated by the available free- CO₂ concentration and not by total dissolved inorganic carbon. Kinetic studies provided support for a CO₂- concentrating mechanism in that the K _0.5[CO₂] (i.e. the CO₂ concentration required for the half-maximal rate of photosynthesis) was substantially lower than the K _m[CO₂] of Rubisco from related taxa, whilst the intracellular carbon pool was at least seven fold greater than the extracellular DIC concentration, for extracellular DIC values 1.0 m m .
It is proposed that when the flux of CO₂ into the cell is insufficient to support the photosynthetic rate at an optimum photon irradiance, the development of CA_ext increases the availability of CO₂ at the plasma membrane. This ensures rapid acclimation to environmental change and provides an explanation for the central role of M. pusilla as a carbon sink in oligotrophic environments.     

LEAF INFECTION OF COTTON BY XANTHOMONAS MALVACEARUM (E.F.SM.) DOWSON

Xanthomonas malvacearum spread more rapidly along vascular tissue than into mesophyll when inoculated to the main veins of susceptible cotton leaves. The extent of spread varied with the concentrations of inocula, tissue age and cotton variety.
Increasing concentrations of inocula accelerated the initial spread of disease.
Bacteria spread more rapidly in young leaves than in old—increasing age greatly decreased disease in the mesophyll. The initial invasion was quicker in young leaves of young plants than in young leaves of old plants.
Three types of behaviour, according to the host's reaction, distinguish Knight's resistance factors: ( a ) where X. malvacearum spread extensively along veins and into mesophyll of plants containing factors B₃ and B₅; ( b ) where it was restricted to the point of inoculation in plants containing B ₄, B₉ and combinations with B _6m; and ( c ) where it spread along veins but not appreciably into mesophyll in varieties containing B ₂ and B ₂ B ₃.
From this and four other different types of tests, factors B ₂ and B ₃ seem to increase mesophyll resistance but only B ₂ gives appreciable vascular resistance. Further, the vascular bundles in varieties with B ₂ seem to be surrounded by an additional 'barrier' which resists X. malvacearum.     

EXPRESSION OF A BEE-VENOM PHOSPHOLIPASE A2 FROM APIS CERANA CERANA IN ESCHERICHIA COLI

Abstract  The venomous phospholipase A₂ (AcPLA₂) coding reading region of the Chinese honeybee ( Apis cerana cerana ), which is composed of 405 bp encoding a mature glycosylated peptide with 134 amino residues was transformed into the expression vector pETblue-1. Then the recombinant vector was introduced into Escherichia coli Tuner (DE3) plac I for expression. Analysis result of SDS-PAGE showed that the expression products had a protein band of about 15kD. Detection of western blot using ant-European honeybee ( Apis mellifera ) phospholipase A₂ (AmPLA₂) polyclonal serum as the first antibody showed that the expression products appeared a special blot same as the native AmPLA₂.The result demonstrated that the AcPLA₂ peptide had been expressed in E. coli and the AcPLA₂ has the similar antigenicity as the AmPLA₂.     

Ozone induced emissions of biogenic VOC from tobacco: relationships between ozone uptake and emission of LOX products

Volatile organic compound (VOC) emissions from tobacco ( Nicotiana tabacum L. var. Bel W3) plants exposed to ozone (O₃) were investigated using proton-transfer-reaction mass-spectrometry (PTR-MS) and gas chromatography mass-spectrometry (GC-MS) to find a quantitative reference for plants' responses to O₃ stress. O₃ exposures to illuminated plants induced post-exposure VOC emission bursts. The lag time for the onset of volatile C₆ emissions produced within the octadecanoid pathway was found to be inversely proportional to O₃ uptake, or more precisely, to the O₃ flux density into the plants. In cases of short O₃ pulses of identical duration the total amount of these emitted C₆ VOC was related to the O₃ flux density into the plants, and not to ozone concentrations or dose–response relationships such as AOT 40 values. Approximately one C₆ product was emitted per five O₃ molecules taken up by the plant. A threshold flux density of O₃ inducing emissions of C₆ products was found to be (1.6 ± 0.7) × 10⁻⁸ mol m⁻² s⁻¹.     

Hydrogen peroxide release from bacterial spores during germination

The release of free H₂O₂ from spores of Clostridium perfringens and Bacillus megaterium during germination has been demonstrated using the scopoletin fluorescence assay. Scopoletin oxidation was markedly inhibited when exogenous catalase was added, and was also influenced by the concentration of spores. H₂O₂ release into the germination medium was observed to parallel the O₂ consumption during germination, suggesting that the H₂O₂ may arise from certain O₂-dependent metabolism associated with initiation of spore germination.     

Agrobacterium tumefaciens-mediated barley transformation

Genetically transformed barley was produced by eco-cultivating immature embryo explants with Agrobacterium tumefaciens carrying a binary vector coding for chimaeric bacterial genes, bar and gus , and selecting for bialaphos-resistant cultures from which plants were regenerated. Integration of both genes was confirmed by gel blot hybridization analysis of DNA from the transformed plants and their progenies. From 1282 embryos, plants were recovered for 54 independently transformed lines, giving a transformation efficiency of 4.2%. Transgene numbers in the different lines ranged from single copy insertion to at least ten copies. Sixteen out of 18 plants grown to maturity were fully fertile. Both marker genes, bar and gus , were expressed and co-segregated in the T₁ progeny plants. In the majority of cases, the genes showed Mendelian segregation predicted for transgene insertion at a single locus. In one family with multiple transgene insertions, molecular analysis of T₁ and T₂ plants suggested that the T-DNA had inserted at two unlinked loci.     

Non-steady state xylem transport of fifteen elements into the tomato leaf as measured by gamma-ray spectroscopy: A model

A model describing the transport of elements through the xylem vessels into the leaf of a red cherry tomato ( Lycopersicon esculentum Mill cv. Tiny Tim) in a non-steady state situation is presented. The model describes the upward movement of ions as a mass-flow of the xylem fluid with dissolved elements, with lateral ion escape represented by a first-order process. The model is fitted to data obtained in an experiment in which 15 elements were applied in a solution to a cut stem part with attached leaf and were measured simultaneously by gamma-ray spectroscopy. The model is in good agreement with the transport into the leaf of K⁺ Na⁺, Rb⁺, Cs⁺, Yb³⁺, Sm³⁺ Zn²⁺, Co²⁺, Cu²⁺, Sb(SO₄)₂ AsO³⁺₄, WO²⁺₄; and Mo₇O⁶⁺₂₄.
Only indirect data could be obtained for Cd²⁺ and La³⁺ because of their apparently high affinity for charged sites in the cell walls and high escape constant, respectively. The escape constants were relatively low for all anions, probably due to the presence of a large number of negative charges in the cell walls.     

Two tertiary branched tetraamines, N4-aminopropylnorspermidine and N4-aminopropylsperimidine, are the major polyamines in thermophilic eubacteria, Thermoleophilum

Abstract Two novel polyamines were found as major polyamines of Thermoleophilum album and Thermoleophilum minutum , which are Gram-negative eubacteria obligate for thermophily and n -alkane substrates. They were identified as tertiary branched tetraamines, N⁴-aminopropylnorspermidine ( tris (3-aminopropyl)amine) [NH₂(CH₂)₃N-((CH₂)₃NH₂)(CH₂)₃NH₂ or N(CH₂CH₂CH₂NH₂)₃] and N⁴-aminopropylspermidine [NH₂(CH₂)₃N((CH₂)₃NH₂)(CH₂)₄NH₂] by high-performance liquid chromatography and gas chromatography-mass spectrometry.     

Genetics in methanogens: transposon insertion mutagenesis of a Methanococcus maripaludis nifH gene.

We designed a transposon insertion mutagenesis system for Methanococcus species and used it to make mutations in and around a nifH gene in Methanococcus maripaludis. The transposon Mudpur was constructed with a gene for puromycin resistance that is expressed and selectable in Methanococcus species. A 15.6-kb nifH region from M. maripaludis cloned in a lambda vector was used as a target for mutagenesis. A series of 19 independent Mudpur insertions spanning the cloned region were produced. Four mutagenized clones in and around nifH were introduced by transformation into M. maripaludis, where each was found to replace wild-type genomic DNA with the corresponding transposon-mutagenized DNA. Wild-type M. maripaludis and a transformant containing a Mudpur insertion upstream of nifH grew on N2 as a nitrogen source. Two transformants with insertions in nifH and one transformant with an insertion downstream of nifH did not grow on N2. The transposon insertion-gene replacement technique should be generally applicable in the methanococci for studying the effects of genetic manipulations in vivo.