Sensitive chemiluminescent detection of digoxigenin-labeled nucleic acids: a fast and simple protocol and its applications.

A fast and simple protocol for the chemiluminescent detection of digoxigenin-labeled nucleic acids with anti-digoxigenin antibody Fab fragments coupled to alkaline phosphatase and 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1.1 (3,7)]decan]-4- yl)phenyl phosphate as substrate is described. The washing and blocking procedure was optimized to yield low background even on positively charged nylon membranes. The sensitivity of the system is equal or better than radioactive methods. Exposure to x-ray or Polaroid film for up to 30 minutes is sufficient for the detection of 70 femtograms of homologous DNA. Human single-copy genes are detected in Southern blots of as low as 0.3 microgram total placental DNA. Blots can be reprobed multiple times very easily. The advantages of the digoxigenin system are high sensitivity, absence of background and ease of reprobing and are illustrated by applications for single-copy gene detection in genomic blots of human DNA, Northern hybridizations to rare mRNA, detection of E. coli genes on blots of genomic digests after pulse field gel electrophoresis, as well as for nonradioactive DNA sequencing blots with digoxigenin-labeled primers.     

Effects of spines and thorns on Australian arid zone herbivores of different body masses

Summary We investigated the effects of thorns and spines on the feeding of 5 herbivore species in arid Australia. The herbivores were the rabbit (Oryctolagus cuniculus), euro kangaroo (Macropus robustus), red kangaroo (Macropus rufus), sheep (Ovis aries), and cattle (Bos taurus). Five woody plants without spines or thorns and 6 woody plants with thorns were included in the study. The spines and thorns were not found to affect the herbivores' rates of feeding (items ingested/min), but they did reduce the herbivores' rates of biomass ingestion (g-dry/item). The reduction in biomass ingested occurred in two ways: at a given diameter, twigs with spines and thorns had less mass than undefended plants, and the herbivores consumed twigs with smaller diameters on plants with spines and thorns. The relative importance of the two ways that twigs with spines and thorns provided less biomass varied with herbivore body mass. Reduced twig mass was more important for small herbivores, while large herbivores selected smaller diameters. The effectiveness of spines and thorns as anti-herbivore defenses did not vary with the evolutionary history of the herbivores (i.e. native vs. introduced). Spines and thorns mainly affected the herbivores' selection of maximum twig sizes (reducing diameter and mass), but the minimum twig sizes selected were also reduced.     

Dry matter production and photosynthetic capacity in Gossypium hirsutum L. under conditions of slightly suboptimum leaf temperatures and high levels of irradiance

Summary Gossypium hirsutum L. var. Delta Pine 61 was cultivated in controlled-environment chambers at 1000–1100 mol photosynthetically active photons m^-2 s^-1 (medium photon flux density) and at 1800–2000 mol photons m^-2 s^-1 (high photon flux density), respectively. Air temperatures ranged from 20° to 34°C during 12-h light periods, whereas during dark periods temperature was 25° C in all experiments. As the leaf temperature decreased from about 33° to 27° C, marked reductions in dry matter production, leaf chlorophyll content and photosynthetic capacity occurred in plants growing under high light conditions, to values far below those in plants growing at 27° C and medium photon flux densities. The results show that slightly suboptimum temperatures, well above the so-called chilling range (0–12° C), greatly reduce dry matter production in cotton when combined with high photon flux densities equivalent to full sunlight.Abbreviations DW dry weight - F _v variable fluorescence yield - F _M maximum fluorescence yield - PFD photon flux density (400–700 nm)     

Cardenolides from Asclepias asperula subsp. Capricornu and A. Viridis

6′-O-(E-4-hydroxycinnamoyl) Desglucouzarin, the first cardenolide containing a cinnamoyl ester moiety, has been isolated from the ethanolic extract of the milkweed, Asclepias asperula. In addition, five known cardenolides were isolated and identified from A. asperula and A. viridis.     

Distribution and immunolocalization of stachyose synthase in Cucumis melo L.

Indirect evidence for the site of stachyose biosynthesis has been provided by determining the occurrence and distribution of stachyose, raffinose and galactinol, the donor of the galactosyl moiety for stachyose synthesis, in Cucumis melo L. cv. Ranjadew. Studies of enzyme activities for the synthesis of these sugars and their distribution in different plant organs and isolates has led to the conclusion that stachyose is synthesized mainly in mature leaves and seeds. Nevertheless, stachyose-synthase activity varied with leaf age, the developmental stage of a plant, the growing season and the plant cultivar used. No stachyose or stachyose-synthase activity could be detected in isolated mesophyll protoplasts and chloroplasts, whereas both were found in a minor-vein-enriched fraction isolated from mature leaves. The conclusion that stachyose biosynthesis is associated with minor veins was confirmed by immunolocalization of the enzyme. Positive specific immunoreactivity of stachyose synthase with polyclonal anti-stachyose-synthase antibodies, labeled with protein A-gold, was detected in intermediary cells of leaf minor veins. The implication of this local synthesis of the main transport sugar for phloem loading in mature leaves of Cucumis melo is discussed.Abbreviation RUBPCase ribulose-1,5-bisphosphate carboxylase This work was supported by Deutsche Forschungsgemeinschaft. The excellent assistance of Ms. B. Müller in preparing the samples for electron microscopy is gratefully acknowledged. The authors thank Professor H.J. Schneider-Poetsch for anti-RuBPCase antibodies.     

Stachyose synthesis in mature leaves of Cucumis melo. Purification and characterization of stachyose synthase (EC 2.4.1.67)

Galactinol: raffinose-6-galactosyltransferase (EC 2.4.1.67), a stachyose synthase, was extracted from mature leaves of Cucumis melo cv. Ranjadew and was purified to homogeneity by (NH₄)₂SO₄ precipitation, ion-exchange chromatography, gel-filtration and non-denaturing polyacrylamide gel electrophoresis. A specific activity of 516 kat · mg^-1 and a 160-fold purification was achieved. The pH optimum of the enzyme reaction was found to be 6.8 in sodium-phosphate buffer, and the temperature optimum 32° C. The purified enzyme was very sensitive towards SH-poisons but its reaction was hardly affected by changes in the ion composition of the assay medium. The two-substrate enzyme was specific for galactinol and raffmose; uridine-diphosphate galactose and p-nitrophenyl--d-galactoside as well as melibiose were not accepted by the purified enzyme. Stachyose synthesis was competitively inhibited by concentrations >4 mM raffinose as well as 2.5 mM galactinol. The K _m values determined under non-saturating conditions were 3.3 mM for raffinose and 7.7 mM for galactinol. Myoinositol was a strong competitive inhibitor with a K _i of 1.8mM. Galactinol was hydrolyzed in the absence of raffinose with a K _m of 0.8 mM. The pure enzyme is a protein with a molecular weight of at least 95 kDa and an isoelectric point of 5.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two subunits of 45 and 50 kDa. Polyclonal antibodies from rabbit were obtained which were specific for the native enzyme but cross-reacted with other proteins separated under denaturing conditions.Abbreviations DEAE diethylaminoethyl - DTT dithiothreitol - FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This work was supported by Deutsche Forschungsgemeinschaft. The gift of galactinol by Dr. T. Schweizer (Nestlé, Switzerland) is gratefully acknowledged.     

Control of plant growth by nitrogen and phosphorus in mesotrophic fens

A fertilization experiment was carried out in 3 mesotrophic fens to investigate whether plant growth in these systems is controlled by the availability of N, P or K. The fens are located in an area with high N inputs from precipitation. They are annually mown in the summer to prevent succession to woodland. Above-ground plant biomass increased significantly upon N fertilization in the two mid-succession fens studied. In the late-succession fen that had been mown for at least 60 years, however, plant biomass increased significantly upon P fertilization. The mowing regime depletes the P pool in the soil, while it keeps N inputs and outputs in balance. A long-term shift occurs from limitation of plant production by N toward limitation by P. Hence, mowing is a suitable management tool to conserve the mesothrophic character of the fens.     

Persistence or loss of preimposed methylation patterns and de novo methylation of foreign DNA integrated in transgenic mice.

In cultured mammalian cells, foreign DNA can be integrated into the host genome. Foreign DNA is frequently de novo methylated in specific patterns with successive cell generations. The sequence-specific methylation of promoter sequences in integrated foreign DNA is associated with the long-term inactivation of eukaryotic genes. We have now extended these experiments to studies on transgenic mice. As in previous work, a construct (pAd2E2AL-CAT) has been used which consists of the late E2A promoter of adenovirus type 2 (Ad2) DNA fused to the prokaryotic gene for chloramphenicol acetyltransferase (CAT). This construct has been integrated in the non-methylated in the 5'-CCGG-3' premethylated form in the genomes of transgenic mice. DNA from various organs was analyzed by HpaII/MspI cleavage to assess the state of methylation in 5'-CCGG-3' sequences. The results demonstrate that the transgenic construct is in general stable. Non-methylated constructs have remained partly non-methylated for four generations or can become de novo methylated at all or most 5'-CCGG-3' sequences in the founder animal. Preimposed patterns of 5'-CCGG-3' methylation have been preserved for up to four generations beyond the founder animal. In the testes of two different founder animals and two F1 males, the transgenic DNA has become demethylated by an unknown mechanism. In all other organs, the transgenic DNA preserves the preimposed 5'-CCGG-3' methylation pattern. In the experiments performed so far we have not observed differences in the transmission of methylation patterns depending on whether the transgene has been maternally or paternally inherited. The 5'-CCGG-3' premethylated transgene does not catalyze CAT activity in several organs, except in one example of the testes of an animal in which the transgenic construct has become demethylated. In contrast, when the nonmethylated construct has been integrated and remained largely non-methylated, CAT activity has been detected in extracts from some of the organs.