Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning

A novel plasmid vector that is able to replicate both in Escherichia coli and in Streptococcus sanguis is described. This 9.2-kb plasmid, designated pVA856, carries Cm^r, Tc^r and Em^r determinants that are expressed in E. coli. Only the Em^r determinant is expressed in S. sanguis. Both the Cm^r and the Tc^r of pVA856 may be insertionally inactivated. This plasmid affords several different cleavage-ligation strategies for cloning in E. coli followed by subsequent introduction of chimeras in to S. sanguis. In addition, we have modified a previously described E. coli-S. sanguis shuttle plasmid [pVA838; Macrina et al., Gene 19 (1982) 345–353], so that it is unable to replicate in S. sanguis. The utility of such a plasmid for cloning and selecting sequences enabling autonomous replication in S. sanguis is demonstrated.     

A SURVEY FOR 1,3,6,7-TETRAHYDROXY-C-GLYCOSYLXANTHONES EMPHASIZING THE “PRIMITIVE” LEPTOSPORANGIATE FERNS AND THEIR ALLIES

A survey for 1,3,6,7-tetrahydroxy-C-glycosylxanthones of representative species within the primitive vascular plants, emphasizing the leptosporangiate ferns, has indicated a limited distribution of these compounds within three leptosporangiate families: Hymenophyllaceae, Aspleniaceae and Marsileaceae. In the Hymenophyllaceae the distribution of these compounds appears to be a useful criterion for segregating species of Mecodium from other species of Hymenophyllum (sensu lato) and suggests that the tubulate vs. the valvate indusial condition may not be an ideal character for separating all species of Hymenophyllum (s.l.) from those of Trichomanes (s.l.). These compounds appear useful for delimiting several species of Elaphoglossum section Pachyglossa and support a relationship among the Aspleniaceae, Athyriaceae, and Elaphoglossaceae. Their presence in Marsilea also raises questions as to the origin of this group of plants.     

Factors affecting chloride conductance in apical membrane vesicles from human placenta

Summary Apical membrane vesicles from human term placenta were isolated using a magnesium precipitation technique, and the purity of the vesicles was assessed morphologically using scanning and transmission electron microscopy, and biochemically, using marker enzymes. The vesicles were found to be morphologically intact and significantly enriched in enzymes associated with apical membranes. ³⁶Cl^– uptake into these vesicles was studied in the presence of an outwardly directed Cl^– gradient. This uptake was found to be time dependent, with an initial rapid uptake tending to peak between 10 and 20 min and thereafter decline. Uptake was found to be voltage dependent since 5 m valinomycin caused a decrease in uptake. The effects of N-phenylanthranilic acid (NPA) and 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) and bumetanide on the initial rate of Cl^– were examined in the presence and absence of 5 m valinomycin. NPA and DIDS inhibited isotope uptake strongly with IC₅₀ values of 0.83±0.35 m and 3.43±0.37 m, respectively, in the absence of valinomycin. Although valinomycin reduced ³⁶Cl^– uptake by about 80% when added before the isotope, DIDS reduced the uptake which remained in a concentration-dependent fashion with an IC₅₀ of 5.6±2.1 m. Under these conditions, NPA was without effect at concentrations below 100 m. Bumetanide was without effect at the concentrations used in the absence of valinomycin. However, following valinomycin pretreatment, bumetanide reduced ³⁶Cl^– uptake significantly at 100 m concentration. Vesicle diameter, as assessed by flow cytometry, did not change under the conditions employed.The effects of some fatty acids were also investigated. Arachidonic acid and linoleic acid inhibited Cl^– uptake with IC₅₀ values of 37.6±14.9 m and 4.59±0.51 m, respectively. Arachidonyl alcohol and elaidic acid were found to be without effect. These studies show that human placental brush border membrane vesicles possess a chloride conductance channel, the activity of which can be measured in the presence of an outwardly directed Cl^– gradient and this channel is sensitive to Cl^– channel inhibitors, especially N-phenylanthranilic acid, and can be inhibited by unsaturated fatty acids such as arachidonic acid and linoleic acid.This work was supported in part by the Cystic Fibrosis Association of Ireland and Eolas, The Irish Science and Technology Agency. The technical assistance of Mr. Cormac O' Connell in the preparation of the electron micrographs and of Mr. Roddy Monks in the flow cytometric analysis is gratefully acknowledged.     

Accumulation of a Polyhydroxyalkanoate Containing Primarily 3-Hydroxydecanoate from Simple Carbohydrate Substrates by Pseudomonas sp. Strain NCIMB 40135

A number of Pseudomonas species have been identified which accumulate a polyhydroxyalkanoate containing mainly 3-hydroxydecanoate monomers from sodium gluconate as the sole carbon source. One of these, Pseudomonas sp. strain NCIMB 40135, was further investigated and shown to accumulate such a polyhydroxyalkanoate from a wide range of carbon sources (C₂ to C₆); however, when supplied with octanoic acid it produced a polyhydroxyalkanoate containing mainly 3-hydroxyoctanoate monomers. Polymer synthesis occurred in batch culture after cessation of growth due to exhaustion of nitrogen. In continuous culture under nitrogen limitation up to 16.9% (wt/wt) polyhydroxyalkanoate was synthesized from glucose as the carbon source. The monomer units are mainly of the R-(−) configuration. Nuclear magnetic resonance studies confirmed the composition of the polymer. Differential scanning calorimetry suggested that the solvent-extracted polymer contained a significant proportion of crystalline material. The weight-average molecular weight of the polymer from glucose-grown cells was 143,000.     

Degradation of extractive-free lignocelluloses by Coriolus versicolor and Poria placenta

Summary The wood-decay fungi Coriolus versicolor, a white-rot fungus, and Poria placenta, a brown-rot fungus, were grown on an extractive-free lignocellulose prepared from quackgrass (Agropyron repens). Their abilities to decompose this lignocellulose were compared to their abilities to decompose softwood (Picea pungens) and hardwood (Acer rubrum) lignocelluloses. The two fungi were grown on malt-extract dampened lignocelluloses at 28°C for up to 12 weeks. Replicate cultures were periodically harvested and lignocellulose decomposition was followed by monitoring substrate weight loss, lignin loss, and carbohydrate loss. Coriolus versicolor decomposed the lignin and carbohydrate components of the grass lignocellulose as efficiently as the softwood and hardwood lignocelluloses. Poria placenta, however, was not an efficient degrader of either lignin or carbohydrate in the grass lignocellulose. Poria placenta readily decomposed carbohydrate components of the softwood lignocellulose but not the hardwood lignocellulose.Paper number 81520 of the Idaho Agricultural Experiment Station     

Kinetics of sugar transport byThiobacillus A2

ApparentK _s andV _max values, for the transport byThiobacillus A2 of¹⁴C-labelled sucrose, hexoses and pentoses, were estimated using flow dialysis and membrane filtration techniques. Transport systems of varying degrees of specificity could be inferred from the data. For most sugars tested including glucose, fructose and arabinose, there was a close correlation between maximum rate of sugar transport and observed growth rate. Differences in transport rate were sufficient to explain slow and fast growth on glucose by wild type and GF strains ofThiobacillus A2.Abbreviations Butyl PBD 2-(4-tert-butylphenyl)-5-(4-biphenylyl)-1,3,4-oxadiazole - Tris tris(hydroxymethyl)-amino-methane - PEP phosphoenolpyruvate