Deletion analysis of sucrose metabolic genes from a Salmonella plasmid cloned in Escherichia coli K12

The sucrose operon from pUR400, a 78-kbp conjugative Salmonella plasmid, was cloned in Escherichia coli K12. The operon was located in a 5.7-kbp SalI restriction fragment and was subcloned, in each of two possible orientations, using the expression vector pUC18. The insert DNA was restriction mapped and duplicate restriction sites in the insert and in the polylinker of the vector were used to create various deletions promoter distal in the operon sequence. Additional deletions were made with the restriction exonuclease Bal31. Cells containing hybrid plasmids with specified deletions lacked the ability to transport sucrose or were constitutive for hydrolase and/or uptake activities. The scrA (enzyme IIScr) and scrR (regulatory) genes resided within 2900-bp SmaI-SalI DNA fragment and were assigned the order scrB, scrA, scrR. An amplified sucrose-inducible gene product, Mr 68,000, was detected only in the membrane fraction from recombinant cells that contained plasmid with the intact operon sequence. This protein represented 11% of the total membrane protein and was resistant to extraction with 0.5 M sodium chloride, 2% Triton X-100, and 0.5% sodium deoxycholate. The protein did not appear to be the product of either the scrA, scrB, or scrR gene and may therefore represent a previously unidentified membrane-bound sucrose protein. A new gene, scrC, is proposed. In addition, the cloned 5.7-kbp SalI and 2.5-kbp SmaI-SalI DNA fragments failed to hybridize to chromosomal DNA from Bacillus subtilis, Streptococcus lactis, Streptococcus mutans, and Lactobacillus acidophilus as well as to DNA from a sucrose plasmid from Salmonella tennessee. However, the probes showed weak homology with a 20-kbp EcoRI restriction fragment from Klebsiella pneumoniae.     

Paternal inheritance of chloroplast DNA in Larix

Restriction enzyme analysis was used to determine the inheritance of chloroplast DNA in conifers. The plant material studied included five individual trees of European larch (Larix decidua Mill.) and Japanese larch (Larix leptolepis Sieb. & Zucc.) and six hybrids from controlled crosses between these species. The chloroplast DNA fragment patterns generated by Bam-HI and Bcl-I were species-specific. Paternal inheritance of chloroplast DNA patterns was found in most Larix crosses. One hybrid showed maternal chloroplast DNA patterns. In addition, two other hybrids had mixed Bam-HI patterns suggesting recombination between maternal and paternal chloroplast DNA. The mechanisms favoring paternal inheritance in conifers are not known. Paternal inheritance of chloroplast DNA is suggested it to be a general phenomenon in conifers.     

Molecular characterization of human platelet glycoproteins IIIa and IIb and the subunits of the latter

Sedimentation equilibrium and low-angle laser-light scattering were used to determine the molar mass of the glycoprotein moieties in the complexes of sodium dodecyl sulphate with the human platelet membrane glycoproteins IIb (GPIIb), IIIa (GPIIIa), and the (GPIIb) and (GPIIb) subunits of GPIIb. The values obtained by both procedures, except those for GPIIb, agree within experimental error with those calculated from their chemical composition: GPIIb (114,000 g mol^-1), GPIIb (22,200 g mol^-1), and GPIIIa (91,500 g mol^-1). The molar mass of GPIIb determined by light scattering (142,000 g mol^-1) and sedimentation equilibrium at different solvent densities (134,000 g mol^-1) also agree, within experimental error, with the values calculated either from its chemical composition (136,500 g mol^-1) or from the sum of the molar masses of its subunits. However the molar mass determined by sedimentation equilibrium at constant solvent density, is consistently underestimated (116,000 g mol^-1).High-performance size-exclusion chromatography in sodium dodecyl sulphate solutions overestimates the molar mass of these glycoproteins and their Stokes radii, and therefore the maximal frictional ratios derived from them.Abbreviations GPIIb glycoprotein IIb - GPIIIa glycoprotein IIIa - GPIIb and GPIIb and subunits of GPIIb, respectively - CM-GPIIb CM-GPIIb, and CM-GPIIIa, totally reduced and carboxymethylated forms of GPIIb, GPIIb, and GPIIIa, respectively - SDS sodium dodecyl sulphate - eosin-ITC eosin-5-isothiocyanate     

Osmotic and cryoprotective effects of glycerol-sucrose solutions on day-3 mouse embryos

The relative volume of Day-3 mouse embryos changed as a linear function of the reciprocal of osmolality [corrected] of non-permeating solutes after 10 min exposure to sucrose and glycerol-sucrose solutions at 20 degrees C. The slope of the linear regression line was less in glycerol-sucrose than in sucrose solutions because glycerol permeation caused re-expansion. Before freezing by direct transfer to -180 degrees C the embryos were placed into glycerol-sucrose in 1-step (1-step equilibration) or first into glycerol and then into glycerol-sucrose (2-step equilibration). Using 2-step equilibration the post-thaw survival rate was substantially higher at 3.0 and 4.0 M-glycerol levels and less dependent on changes in the sucrose concentration within the range of 0.125 to 1.0 M than with 1-step equilibration. Under optimal conditions 90-95% of rapidly frozen embryos developed to blastocysts in vitro and 30% into live young in vivo. It is suggested that the cryoprotective role of glycerol is due to its ability to reduce osmotic pressure differences between the extra and intracellular spaces during rapid freezing of embryos.     

Large Alterations in Ganglioside and Neutral Glycosphingolipid Patterns in Brains from Cases with Infantile Neuronal Ceroid Lipofuscinosis/Polyunsaturated Fatty Acid Lipidosis

Lipid composition was studied on cerebral tissue from nine children who had died of a progressive encephalopathy called the infantile form of neuronal ceroid lipofuscinosis (INCL) or polyunsaturated fatty acid lipidosis (PFAL). In the terminal stage of the disease, the concentrations of all lipid classes were found to be significantly reduced in the cerebral and cerebellar cortex and white matter. The concentration of gangliosides of the cerebral cortex was 15% and that of cerebrosides (galactosylceramide) in white matter 0.2-5% of the normal values for the children's ages. The reduction of gangliosides mainly affected those of the gangliotetraose series, particularly GD1a. The fatty acids of the linolenic acid series were strongly reduced in ethanolamine and serine phosphoglycerides. A very large increase up to 100-fold of oligoglycosphingolipids of the globo series and two fucose-containing lipids of the neolacto series was found in the forebrain of the three advanced cases examined. The brain tissue also contained very high concentrations of mono-, di-, and trisialogangliosides of the lacto and neolacto series, gangliosides with type 1 chain dominating. The structures of the gangliosides were tentatively identified by gas chromatography-mass spectrometry and monoclonal antibodies with carefully determined epitope specificity. The gangliosides and neutral glycosphingolipids had very similar fatty acid composition, consisting of about 40% stearic acid and 40% C24-acids.     

Covalent labeling of opioid receptors with 3H-D-Ala2-Leu5-enkephalin chloromethyl ketone. I. Binding characteristics in rat brain membranes

The chloromethyl ketone derivative of D-Ala2-Leu5-enkephalin was synthesized in a radioactive form, and the resulting compound (3H-DALECK) was used to label opioid receptors. 3H-DALECK binds with high affinity, specificity and saturability to rat brain membranes. The number of sites labeled is 130 fmoles/mg protein. Unlabeled opioids inhibited the binding of 3H-DALECK; etorphine and DAGO being most potent. A 10-fold preference for mu sites over delta was seen in site-specific competition experiments; while DALECK displayed low affinity for kappa sites of rat brain. DALECK irreversibly blocked a certain population of sites. Approximately 40% of 3H-DALECK binding at 15 min, and 60% at 60 min association time did not dissociate in the presence of a large excess of unlabeled DALECK and was resistant to washing. Autoradiography performed after SDS-PAGE revealed specific alkylation of proteins with molecular weight of 74, 65, 56, 43 and 34 kD. These results demonstrate the applicability of using 3H-DALECK to covalently label opioid receptors.     

Comparison of gas chromatography-mass spectrometry,radioimmunoassay and bioassay for the quantification of gibberellin A9 in norway spruce (Picea abies (L.) Karst.)

Quantitative estimates of gibberellin A₉ in Norway spruce extracts obtained by gas chromatography-mass spectrometry, radioimmunoassay (RIA_ and bioassay were compared after successive purifications of the extracts. The extracts were assayed in several dilutions with and without the addition of standard gibberellin A₉, thus showing the effect of extract components on the response of the assays. Radioimmunoassay produced estimates comparable to gas chromatography-mass spectrometry after one purification step by high-performance liquid chromatography. Extracts purified by polyvinylpyrrolidone-column chromatography and solvent partitioning but not high-performance liquid chromatography resulted in inaccurate RIA estimates. The performance of the RIA could be monitored by logit-log transformations of the standard curve and extract dilution curve and by calculating the slope of the standard addition curve. It was, however, not possible to correct for the interference caused by extract components by the standard addition procedure. Quantifications by Tan-ginbozu dwarf-rice bioassay were accurate, but a large and unpredictable variation makes it unsuitable for quantitative determinations.Abbreviations FW fresh weight - GA₉ gibberellin A₉ - GA₉–Me methylated GA₉ - GC-MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - MID multiple-ion detection - RIA radioimmunoassay     

Effects of glyphosate on phenolic metabolism in yellow nutsedge leaves

The action of the herbicide glyphosate [N-(phosphonomethyl)-glycine] on phenolic metabolism and phenylalanine ammonia lyase (PAL; EC 4.3.1.5) activity was investigated in yellow nutsedge (Cyperus esculentus L.). Glyphosate caused significant increases in the amount of total soluble hydroxyphenolics in the three fractions studied (neutral, acid and residual). Qualitative and quantitative differences in relation to these fractions and the amount of applied glyphosate were observed. Most of the phenolic compounds which increased after glyphosate treatment were benzoic acids (gentisic. p -OH-benzoic, salicylic and vanillic). Gentisic acid showed the greatest increase in neutral and acid fractions, being twenty- and four-fold, respectively, of the amount found in the control. PAL activity was not affected by the lowest doses of glyphosate (10−⁴and 10−³ M) , but a dramatic decrease in PAL activity was observed after 10−² M treatment. These findings, together with the low levels of cinnamic acids measured in treated yellow nutsedge plants, suggest that PAL activity is only marginally involved in glyphosate action. Since the herbicidal action probably takes place at 5-enol-pyruvylshikimate-3-P synthase (EC 2.5.1.19), an alternative pathway to PAL in phenolic biosynthesis should be activated yielding benzoic acids.     

Hormone directed sucrose transport during fruit set induced by gibberellins in Pisum sativum

A new system has been developed to study hormone-directed transport in intact plants during parthenocarpic fruit set induced by gibberellins. Gibberellic acid (GA₃) and gibberellin A₁ (GA₁) applied to unpollinated ovaries of pea ( Pisum sativum L. cv. Alaska) promoted sucrose transport from the leaf to the site of hormone application. In vivo experiments showed an early (30 min) accumulation of [¹⁴C]-sucrose in ovaries of pea stimulated by gibberellins. This activation of sucrose transport appears to be mediated by gibberellins (GA₁, GA₃), increasing both loading of phloem with sucrose in the leaf (source) and sucrose unloading in the ovary (sink). The ability of pea tissue segments to take up sucrose in vitro was not affected by the hormonal treatment.