Limited Tryptic Proteolysis of the Benzodiazepine Binding Proteins in Different Species Reveals Structural Homologies

Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.     

Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.

The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of approximately 1%. Site-directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem-loop structure. Part of this stem-loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.     

The intron 7 donor splice site transition: a second Tay-Sachs disease mutation in French Canada

Mutations at the hexosaminidase A (HEXA) gene which cause Tay-Sachs disease (TSD) have elevated frequency in the Ashkenazi Jewish and French-Canadian populations. We report a novel TSD allele in the French-Canadian population associated with the infantile form of the disease. The mutation, a GA transition at the +1 position of intron 7, abolishes the donor splice site. Cultured human fibroblasts from a compound heterozygote for this transition (and for a deletion mutation) produce no detectable HEXA mRNA. The intron 7+1 mutation occurs in the base adjacent to the site of the adult-onset TSD mutation (G805A). In both mutations a restriction site for the endonuclease EcoRII is abolished. Unambiguous diagnosis, therefore, requires allele-specific oligonucleotide hybridization to distinguish between these two mutant alleles. The intron 7+1 mutation has been detected in three unrelated families. Obligate heterozygotes for the intron 7+1 mutation were born in the Saguenay-Lac-St-Jean region of Quebec. The most recent ancestors common to obligate carriers of this mutation were from the Charlevoix region of the province of Quebec. This mutation thus has a different geographic centre of diffusion and is probably less common than the exon 1 deletion TSD mutation in French Canadians. Neither mutation has been detected in France, the ancestral homeland of French Canada.     

A minority of 46,XX true hermaphrodites are positive for the Y-DNA sequence including SRY

Summary A total of 30 cases of 46,XX true hermaphroditism was analysed for Y-DNA sequences including the recently cloned gene for male testis-determination SRY. In 3 cases, a portion of the Y chromosome including SRY was present and, in 2 cases, was localised, to Xp22 by in situ hybridisation. Since previous studies have shown that the majority of XX males are generated by an X-Y chromosomal interchange, the Xp22 position of the Yp material suggests that certain cases of hermaphroditism can arise by the same meiotic event. The phenotype in the 3 SRY-positive cases may be caused by X-inactivation resulting in somatic mosaicism of testis-determining factor expression giving rise to both testicular and ovarian tissues. Autosomal or X-linked mutation(s) elsewhere in the sex-determining pathway may explain the phenotype observed in the remaining 27 SRY-negative cases.     

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.     

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.