Arabidopsis glycosyltransferases as biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides

Regioselectivity of glycosyltransferases offers an important means to overcome the limitations of chemical synthesis of small molecule glycosides. In this study we explore a large multigene family of UDP-glucose:glycosyltransferases of Arabidopsis for their potential as novel biocatalysts for in vitro synthesis and whole-cell catalysis. We used quercetin as a substrate for this study because the flavonol and its glycosides have important medicinal properties and the metabolite provides a complex structure for regioselective glucosylation. We analyzed the activity of 91 recombinant enzymes for in vitro activity toward quercetin and discovered 29 that are capable of glucosylating the substrate. We demonstrate the first enzymic synthesis of a range of glucosides in vitro, including the 3-O-, 7-O-, 3'-O-, and 4'-O-monoglucosides, 3,7-di-O-glucoside, and 7,3'-di-O-glucoside. We also show that the regioselectivity of glucosylation can be maintained when the enzymes are used as whole-cell biocatalysts in Escherichia coli.     

Marker rescue of adeno-associated virus (AAV) capsid mutants: a novel approach for chimeric AAV production

Marker rescue, the restoration of gene function by replacement of a defective gene with a normal one by recombination, has been utilized to produce novel adeno-associated virus (AAV) vectors. AAV serotype 2 (AAV2) clones containing wild-type terminal repeats, an intact rep gene, and a mutated cap gene, served as the template for marker rescue. When transfected alone in 293 cells, these AAV2 mutant plasmids produced noninfectious AAV virions that could not bind heparin sulfate after infection with adenovirus dl309 helper virus. However, the mutation in the cap gene was corrected after cotransfection with AAV serotype 3 (AAV3) capsid DNA fragments, resulting in the production of AAV2/AAV3 chimeric viruses. The cap genes from several independent marker rescue experiments were PCR amplified, cloned, and then sequenced. Sequencing results confirmed not only that homologous recombination occurred but, more importantly, that a mixed population of AAV chimeras carrying 16 to 2,200 bp throughout different regions of the type 3 cap gene were generated in a single marker rescue experiment. A 100% correlation was observed between infectivity and the ability of the chimeric virus to bind heparin sulfate. In addition, many of the AAV2/AAV3 chimeras examined exhibited differences at both the nucleotide and amino acid levels, suggesting that these chimeras may also exhibit unique infectious properties. Furthermore, AAV helper plasmids containing these chimeric cap genes were able to function in the triple transfection method to generate recombinant AAV. Together, the results suggest that DNA from other AAV serotypes can rescue AAV capsid mutants and that marker rescue may be a powerful, yet simple, technique to map, as well as develop, chimeric AAV capsids that display different serotype-specific properties.     

Evolution of substrate recognition across a multigene family of glycosyltransferases in Arabidopsis

The complete sequence of the Arabidopsis genome enables definitive characterization of multigene families and analysis of their phylogenetic relationships. Using a consensus sequence previously defined for glycosyltransferases that use small-molecular-weight acceptors, 107 gene sequences were identified in the Arabidopsis genome and used to construct a phylogenetic tree. Screening recombinant proteins for their catalytic activities in vitro has revealed enzymes active toward physiologically important substrates, including hormones and secondary metabolites. The aim of this study has been to use the phylogenetic relationships across the entire family to explore the evolution of substrate recognition and regioselectivity of glucosylation. Hydroxycoumarins have been used as the model substrates for the analysis in which 90 sequences have been assayed and 48 sequences shown to recognize these compounds. The study has revealed activity in 6 of the 14 phylogenetic groups of the multigene family, suggesting that basic features of substrate recognition are retained across substantial evolutionary periods.     

Plants in a cold climate

Plants are able to survive prolonged exposure to sub-zero temperatures; this ability is enhanced by pre-exposure to low, but above-zero temperatures. This process, known as cold acclimation, is briefly reviewed from the perception of cold, through transduction of the low-temperature signal to functional analysis of cold-induced gene products. The stresses that freezing of apoplastic water imposes on plant cells is considered and what is understood about the mechanisms that plants use to combat those stresses discussed, with particular emphasis on the role of the extracellular matrix.     

Metschnikowia arizonensis and Metschnikowia dekortorum,two new large-spored yeast species associated with floricolous beetles

Two new haplontic heterothallic species of Metschnikowia were discovered in flowers and associated beetles. Metschnikowia arizonensis was recovered from flowers of cholla cactus (Opuntia echinocarpa) and a specimen of Carpophilus sp. (Coleoptera: Nitidulidae) found in these flowers, in Arizona. Metschnikowia dekortorum was isolated in specimens of the nitidulid beetle Conotelus sp. captured in flowers of two species of Ipomoea in northwestern Guanacaste Province, Costa Rica. The sexual cycle of these yeasts is typical of the large-spored Metschnikowia species, but the asci and spores are intermediate in size between these and other members of the genus. The physiology is consistent with that of most Metschnikowia species except that both species fail to utilize lysine as sole nitrogen source. Also, M. arizonensis utilizes fewer carbon compounds than most species and exhibits considerable variability among strains at this level. Partial ribosomal DNA large-subunit (D1/D2) sequences suggest that M. arizonensis and M. dekortorum are moderately related sister species whose positions are intermediate between the large-spored species Metschnikowia and Metschnikowia hibisci. The type cultures are: M. arizonensis, strains UWO(PS)99-103.3.1=CBS 9064=NRRL Y-27427 (h(+), holotype) and UWO(PS)99-103.4=CBS 9065=NRRL Y-27428 (h(-), isotype); and M. dekortorum, strains UWO(PS)01-142b3=CBS 9063=NRRL Y-27429 (h(+), holotype) and UWO(PS)01-138a3=CBS 9062=NRRL Y-27430 (h(-), isotype).     

Soil warming and trace gas fluxes: experimental design and preliminary flux results

We conducted several experiments to determine a procedure for uniformly warming soil 5° C above ambient using a buried heating cable. These experiments produced a successful design that could: 1) maintain a temperature difference of 5° C over a wide range of environmental conditions; 2) reduce inter-cable temperture variability to ca. 1.5° C; 3) maintain a temperature difference of 5° C near the edges of the plot; and 4) respond rapidly to changes in the environment. In addition, this design required electrical power only 42% of the time. Preliminary measurements indicate that heating increased CO₂ emission by a factor of ca. 1.6 and decreased the C concentration in the O soil horizon by as much as 36%. In addition, warming the soil accelerated the emergence and early growth of the wild lily of the valley (Maianthemum canadense Desf.). The relationship between CO₂ flux and soil temperature derived from our soil warming experiment was consistent with data from other hardwood forests around the world. Since the other hardwood forests were warmed naturally, it appears that for soil respiration, warming the soil with buried heating cables differs little from natural, aboveground warming. By warming soil beyond the range of natural variability, a multi-site, long-term soil warming experiment may be valuable in helping us understand how ecosystems will respond to global warming.     

Interactions between platelet-surface glycoproteins. Identification of glycoproteins capable of binding to platelet surfaces.

1. Platelets have been isolated from plasma and their surface glycoconjugates radioactively-labelled using galactose oxidase and NaB3H4. 2. Conditions have been defined for optimal labelling of glycoproteins and a membrane fraction enriched in plasma membrane has been prepared and characterized by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. Desialylated glycoproteins that act as receptors to peanut agglutinin and lentil lectin have been purified from a detergent extract of plasma membrane. 4. Two glycosylated polypeptides that are able to bind to the surfaces of platelets have been identified and some characteristics of the binding have been investigated.     

Asian (Taiwan) Taenia: species or strain?

Recent studies on the epidemoiological pattern of taenisis in Southeast Asia have indicated the existence of a third form of human Taenia which is distinguishable from Taenia saginata and T. solium. Don McManus and Josephine Bowles here review how new genetic evidence supports earlier conclusions that the Asian Taenia is a distinct entity but is closely related T. saginata, and suggests its taxonomic classification as a subspecies or strain of T. saginata is more appropriate than formal designation as a new species.     

Distribution and characterization of recrystallization inhibitor activity in plant and lichen species from the UK and maritime Antarctic

Extracts from a range of evolutionarily diverse plant and lichen species from the UK and maritime Antarctic have been assayed for inhibition of ice recrystallization. Approximately 25% of overwintering UK species and all Antarctic species exhibited antifreeze activity when exposed to low temperature. Preliminary characterization of the active extracts has demonstrated that the molecules co-opted to antifreeze activity by different species are biochemically diverse.