Detection of microgram quantities of carrier ampholytes in electrofocused proteins by thin-layer chromatography

A rapid and sensitive method for the detection of carrier ampholyte contamination in electrofocused proteins is described. Samples containing proteins and carrier ampholytes were applied to cellulose thin-layer chromatographic sheets and developed in 10% trichloroacetic acid. Proteins and large-molecular-weight carrier ampholytes were precipitated at the origin while 10% trichloroacetic acid-soluble carrier ampholytes migrated as a diffuse ninhydrin (nitrogen)-positive area at an R_f greater than 0.50. We found that 1.25 μg of carrier ampholytes contained enough 10% trichloroacetic acid-soluble components to be detected by thinlayer chromatography. Using this assay, we investigated techniques designed to remove carrier ampholytes from an electrofocused protein. Removal of large-molecular-weight components from carrier ampholytes by dialysis through a 3500 M_r cutoff membrane did not facilitate separation of carrier ampholytes from streptococcal pyrogenic exotoxin type C by dialysis or gel chromatography. Also, this protein binds irreversibly to mixed-bed ion-exchange resin. The best method for separating carrier ampholytes from streptococcal pyrogenic exotoxin type C was by electrodialysis at pH 4.0. Following electrodialysis, estimated carrier ampholyte contamination in this protein was less than 1 part in 500 parts (by weight).     

Function of an internal bacteriophage T7 core during assembly of a T7 procapsid.

A DNA-free, proteinaceous procapsid of bacteriophage T7 (capsid I) has been shown in previous studies to consist of an external, spherical shell (envelope) and an internal, cylindrical core with fibrous projections that connect the core to the envelope. To determine the role of the core in assembly of the envelope of capsid I, the kinetics of appearance of capsid I and possible intermediates in capsid I assembly (AG particles) were determined in the presence and absence of the core. For obtaining these data, agarose gel electrophoresis was used and appeared to be a technique more accurate and efficient than techniques used for obtaining similar data in the past. The results of these experiments were: (i) in the presence of the core, AG particles behaved kinetically as intermediates in the assembly of capsid I; (ii) in the absence of the core, assembly of capsid I terminated prematurely and AG particles accumulated. These and other data have been interpreted by assuming that: AG particles are breakdown products of precursors of capsid I; these precursors have uncorrected errors in the assembly of their envelope; and a function of the core is to correct these errors.     

Isolation of an Hfr donor of Pseudomonas aeruginosa PAO by insertion of the plasmid RP1 into the tryptophan synthase gene

Summary A derivative of the IncP-1 plasmid RP1, temperature-sensitive for maintenance, was inserted into the Pseudomonas aeruginosa chromosome by selection for a plasmid marker (carbenicillin resistance) at nonppermissive temperature. In one strain, PAO 1000, the plasmid was stably integrated in the trpA, B gene cluster mapped at 27 min, as shown by the following evidence. (i) Trp⁺ transductants lost all plasmid markers. (ii) Cleared lysates of PAO 1000 showed no plasmid band typical of the autonomous RP1 in agarose gel electrophoresis. (iii) No transfer of carbenicillin resistance by PAO 1000 was detectable. (iv) PAO 1000 mobilised the chromosome from an origin at, or very near, the plasmid insertion site with high frequency (recovery of proximal markers 10^–3 per donor). Matings on the plate with and without interruption of conjugation showed that chromosome transfer was unidirectional. (v) Recombinants from PAO 1000-mediated crosses did not inherit plasmid markers or the trpA, B mutation. A derivative of PAO 1000 was obtained which had lost the Hfr property and all plasmid markers except carbenicillin resistance. This strain (PAO 1001), when carrying the autonomous RP1 plasmid, was capable of unidirectional chromosome mobilisation like PAO 1000, but with 50-fold lower efficiency. We propose that integration of the temperature-sensitive RP1 plasmid in PAO 1000 occurred via transposition of Tnl, the element specifying carbenicillin resistance.     

Systematic variation in amino acid compositions of grass caryopses

Variation in amino acid patterns of 121 species (72 genera) of grass caryopses is extensively consistent with taxonomic groupings. The patterns of pooids and chloridoids are distinguishable from one another and from those of eu-panicoids and andropogonoids; the bamboos, Oryza, Stipeae, Ehrharta and Microlaena, which share certain morphological and anatomical features, also share a characteristic amino acid profile, while profiles of danthonoioids, Triodia and Aristida are clearly non-pooid. Caryopsis amino acid patterns vary independently of photosynthetic pathway. Embryos from taxonomically diverse genera all show very similar amino acid profiles, which differ strikingly from those of the endosperms, and the amino acid patterns of whole caryopses are dominated by their endosperms, which are responsible for the taxonomic variation. ‘Chemical scores’ of the caryopsis proteins, but not total protein contents, correlate to some extent with taxonomic groupings.     

Unsaturated fatty acid but not ergosterol is essential for high ethanol production in Saccharomyces

Summary The membrane lipid composition of Saccharomyces was manipulated by growing cells anaerobically with or without ergosterol and unsaturated fatty acid. Cells low in ergosterol but enriched in unsaturated fatty acid residues on membrane phospholipids produced high concentrations, 13–15.5% w/v, of ethanol at substrate conversion efficiencies of around 90%.     

Structure and activity of phosphoglycerate mutase

The structure of yeast phosphoglycerate mutase determined by X-ray crystallographic and amino acid sequence studies has been interpreted in terms of the chemical, kinetic and mechanistic observations made on this enzyme. There are two histidine residues at the active site, with imidazole groups almost parallel to each other and approximately 0.4 nm apart, positioned close to the 2 and 3 positions of the substrate. The simplest interpretation of the available information suggests that a ping-pong type mechanism operates in which at least one of these histidine residues participates in the phosphoryl transfer reaction. The flexible C-terminal region also plays an important role in the enzymic reaction.     

Pectate distribution and esterification in Dubautia leaves and soybean nodules,studied with a fluorescent hybridization probe

Carbohydrate-hybridization probes (Vreeland and Laetsch, 1989, Planta (177, 423–434) were used to localize the homogalacturonan (pectate) component of pectins in the cell walls of leaves and soybean root nodules. Leaves of two species of the dicotyledon Dubautia were compared; these species contain much pectin but differ in their tissue water relations with respect to their cell-wall properties. Maturation of the primary cell walls in nodules was studied in the Bradyrhizobium japonicum-Glycine max symbiosis. Probe labelling was based on the divalent-cation-mediated association between pectate in tissue sections and fluorescein-conjugated pectate fragments. Pectate was also labelled by mixed-dimer formation with fluorescent polyguluronate derived from alginate. The specificity of the probe for unesterified polygalacturonate was indicated by increased cell-wall labelling after chemical or enzymatic deesterification of tissue sections, in contrast to elimination of labelling by chemical esterification. Postfixation of tissue sections improved retention of soluble pectate. Pectate differences were found in the leaves among cell types, in degree of esterification, and between plant species. The cell walls of soybean nodules were strongly labelled by the pectate probe in nodules one week and three weeks after infection. Pectate was more highly esterified in the central infected zone than in the surrouding cortex. Within the infected zone, walls of uninfected cells and infected cells were similarly labelled by the pectate probe. The results indicate that the pectate molecular probe provides detailed information on pectate distribution at the cellular level for investigations of cell-wall structure, development and physiology.Abbreviations EDTA ethylenedinitrilotetraacetic acid (ethylenediaminetetraacetic acid) - NMR nuclear magnetic resonance spectroscopy - TTB 1,3,5-triazido-2,4,6-trinitrobenene     

Modulation of cytoskeletal organization during insect follicle cell morphogenesis

Follicle cell morphogenesis during Rhodnius oogenesis involves extensive changes in lateral follicle cell shape, creating a patent epithelium. Cytoskeletal elements are involved in this cell shape change as assessed by investigating the relative abundance, orientation and dynamics of the follicle cell microtubule and microfilament cytoskeleton. Anti-tubulin immunofluorescence and transmission electron microscopy revealed the cytoskeletal organization from pre-follicular to post-vitellogenic follicle stages. A well-developed cylindrical arrangement of longitudinally orientated microtubules is present beneath the plasmalemma of the non-patent pre-vitellogenic and apical vitellogenic follicle cells. In contrast patent lateral vitellogenic follicle cells contain a dispersed distribution of microtubules in both longitudinal and cross-sectional planes. Prominent microfilament bands are not abundant in the pre-vitellogenic or apical vitellogenic follicle cells. The lateral vitellogenic follicle cells do however contain a prominent band of microfilaments in the subplasmalemmal area and in the projections connecting to adjacent cells and the apical microvilli. The changes in cytoskeletal arrangement in lateral follicle cells during vitellogenesis emphasize a third essential component, in addition to juvenile hormone stimulated [NA(+)K(+)] ATPase cell shrinkage, and cell junctional modulation, for the formation of a patent follicle cell epithelium in Rhodnius.