Environmental risk assessments for transgenic crops producing output trait enzymes

The environmental risks from cultivating crops producing output trait enzymes can be rigorously assessed by testing conservative risk hypotheses of no harm to endpoints such as the abundance of wildlife, crop yield and the rate of degradation of crop residues in soil. These hypotheses can be tested with data from many sources, including evaluations of the agronomic performance and nutritional quality of the crop made during product development, and information from the scientific literature on the mode-of-action, taxonomic distribution and environmental fate of the enzyme. Few, if any, specific ecotoxicology or environmental fate studies are needed. The effective use of existing data means that regulatory decision-making, to which an environmental risk assessment provides essential information, is not unnecessarily complicated by evaluation of large amounts of new data that provide negligible improvement in the characterization of risk, and that may delay environmental benefits offered by transgenic crops containing output trait enzymes

Crystal structures of leucyl/phenylalanyl-tRNA-protein transferase and its complex with an aminoacyl-tRNA analog

Eubacterial leucyl/phenylalanyl-tRNA protein transferase (L/F-transferase), encoded by the aat gene, conjugates leucine or phenylalanine to the N-terminal Arg or Lys residue of proteins, using Leu-tRNA(Leu) or Phe-tRNA(Phe) as a substrate. The resulting N-terminal Leu or Phe acts as a degradation signal for the ClpS-ClpAP-mediated N-end rule protein degradation pathway. Here, we present the crystal structures of Escherichia coli L/F-transferase and its complex with an aminoacyl-tRNA analog, puromycin. The C-terminal domain of L/F-transferase consists of the GCN5-related N-acetyltransferase fold, commonly observed in the acetyltransferase superfamily. The p-methoxybenzyl group of puromycin, corresponding to the side chain of Leu or Phe of Leu-tRNA(Leu) or Phe-tRNA(Phe), is accommodated in a highly hydrophobic pocket, with a shape and size suitable for hydrophobic amino-acid residues lacking a branched β-carbon, such as leucine and phenylalanine. Structure-based mutagenesis of L/F-transferase revealed its substrate specificity. Furthermore, we present a model of the L/F-transferase complex with tRNA and substrate proteins bearing an N-terminal Arg or Lys

Inheritance of glabrous plants in Helianthemum oelandicum var. oelandicum and spatial patterns of allele frequencies in local populations

Helianthemum oelandicum var. oelandicum is an endemic taxon on the Baltic island of Öland, SE Sweden. Plants can be classified into two morphs: the bristled morph (with bristles and with or without scattered stellate hairs) and the glabrous morph (without bristles and stellate hairs). In crosses between plants assumed to be homozygous for the trait that characterises the phenotypes of the two morphs, offspring in F1 could not be distinguished from the bristled morph. Segregation in F2 did not deviate from the expected 3:1 ratio (bristled morphs/glabrous morphs), indicating one major gene with a dominant allele for the phenotype of the bristled morph and a recessive allele for the phenotype of the glabrous morph. Besides the Mendelian inheritance of presence/absence of hairs, the density of hairs appeared to be further modified by quantitative genes. The frequency of the recessive allele for the phenotype of the glabrous morph varied among local populations and showed a geographical structure, both on local and regional scales. Possible mechanisms behind the spatial variation in indumentum are discussed

Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity

Abstract O-sulfotransferases (OSTs) are critical enzymes in the cellular biosynthesis of the biologically and phar-macologically important heparan sulfate and heparin. Re-cently, these enzymes have been cloned and expressed in bacteria for application in the chemoenzymatic synthesis of glycosaminoglycan-based drugs. OST activity assays have largely relied on the use of radioisotopic methods using [35S] 3′-phosphoadenosine-5′-phosphosulfate and scintillation counting. Herein, we examine alternative as-says that are more compatible with a biomanufacturing environment. A high throughput microtiter-based approach is reported that relies on a coupled bienzymic colorimetric assay for heparan sulfate and heparin OSTs acting on polysaccharide substrates using arylsulfotransferase-IV and p-nitrophenylsulfate as a sacrificial sulfogroup donor. A second liquid chromatography-mass spectrometric assay, for heparan sulfate and heparin OSTs acting on structurally defined oligosaccharide substrates, is also reported that pro-vides additional information on the number and positions of the transferred sulfo groups within the product. Together, these assays allow quantitative and mechanistic information to be obtained on OSTs that act on heparan sulfate and heparin precursors