Maintenance of the induced hypomethylated state of tobacco nuclear repetitive DNA sequences in the course of protoplast and plant regeneration

Previously, we established experimental conditions allowing us to induce hypomethylation of tandem arrays of highly repetitive DNA sequences in the Nicotiana tabacum L. nuclear genome (M. Bezdk et al. 1991, Planta 184, 487–490). In this paper, we demonstrate that loci containing the highly repetitive sequences of the HRS60 family can maintain the induced hypomethylated state through protoplast regeneration, non-differentiated callus growth, and plant regeneration. The hypomethylation, induced with 5-azacytidine and monitored on these sequences, did not substantially alter the capacity of calli to produce plants. It can be concluded that, in contradistinction of multiple copies of transgenes or ectopic genes which are usually recognized as methylation targets, endogenous tandem repeats, such as the HRS60, present at 10⁵ copies in the genome, can escape de-novo methylation.Abbreviation AzaC 5-azacytidine This project was supported by the Grant Agency of the Academy of Sciences of the Czech Republic. We thank Ms. Libue Jedliková, Ms. Hana Suchánková, and Ms. Emilie Koudelková for technical assistance.     

Activities of a Mechanosensitive Ion Channel in anE. Coli Mutant Lacking the Major Lipoprotein

Summary The activity of the mechanosensitive (MS) ion channels in membrane patches, excised fromE. coli spheroplasts, was analyzed using the patch-clamp technique. Outer membranes from a mutant lacking the major lipoprotein (Lpp) and its wildtype parent were examined. The MS-channel activities in the wild-type membrane rarely revealed substates at the time resolution used. These channels showed a stretch sensitivity indicated by the IISP (the suction for ane-fold increase in channel open probability) of 4.9 mm Hg suction. The MS-channel activities oflpp included a prominent substate and showed a weaker mechano-sensitivity with an 1/S _p of 10.0 mm Hg. Whereas small amphipaths (chlorpromazine, trinitrophenol) or a larger amphipath (lysolecithin) all activated the MS channel in the wild-type membrane under minimal suction, only the larger lysolecithin could activate the MS channel in thelpp membranes. After lysolecithin addition, thelpp membrane became more effective in transmitting the stretch force to the MS channel, as indicated by a steepening of the Boltzmann curve. We discuss one interpretation of these results, in which the major lipoprotein serves as a natural amphipath inserted in the inner monolayer and the loss of this natural amphipath makes the bilayer less able to transmit the gating force.     

Getting to the inside of cells using metabolic control analysis

Metabolic control analysis can relate control properties of an intact system to kinetic properties (elasticity coefficients) of the enzymes within that system. The method formulating the former as matrix inverse of the latter is elaborated here for the general case and founded in standard metabolic control theory. Then a method is developed that accomplishes the reverse: it is shown that a matrix containing all elasticity coefficients and information concerning the pathway structure equals the inverse of a matrix containing flux and concentration control coefficients. As a consequence, by measuring the control properties of an intact system, one is able to deduce its in situ pathway structure and enzyme kinetic properties: This solves the ever-present question of whether the kinetic properties of enzymes in their isolated state differ from those under the conditions prevailing in the cell.     

The Product of glnL Is Not Essential for Regulation of Bacterial Nitrogen Assimilation

The glnL product is not necessary for the control of expression of glnA and other nitrogen-regulated genes, but it presumably communicates reduntant information on the availability of ammonia from an ammonia-sensitive system consisting of the products of glnB and glnD to the regulatory products of glnF and glnG.     

Influence of 4′-6-diamidino-2-phenylindole on the secondary structure and template activities of DNA and polydeoxynucleotides

Summary The interaction between 4-6-Diamidino-2-Phenylindole-hydrochloride (DAPI) and a variety of DNAs and synthetic polydeoxynucleotides was investigated in order to delineate the nucleic acid structural features necessary for binding. The spectra of DAPI-DNA complexes, measured at various DAPI-DNA molar ratios (r), are hypochromic relative to DNA in the region of its maximum absorption. All the curves pass through an isosbestic point at 268 nm. A new maxima appears in the region of 380–392 nm for DAPI-DNA complexes. The magnitude of the peaks in the region are directly proportional to the amount of drug present in the complex.Studies with various DNA types and synthetic polydeoxynucleotides indicate that the drug preferentially binds to dAT-rich regions of DNA. This was also confirmed by enzymatic studies. The inhibition of template action by DAPI in a purified DNA-polymerase reaction was dependent on the dAT-content of the template. The implication of these data to explain a selective binding of DAPI to mitochondrial DNA have been discussed.     

Electrically induced Ca2+ transport across the membrane of Paramecium caudatum measured by means of flow-through technique

Transmembrane calcium fluxes related to excitation were studied in Paramecium caudatum. Radioactive (⁴⁵Ca²⁺) or inactive solution was flowed through a dense suspension of unlabelled or labelled cells, and radioactivity was monitored in the solution. The organisms were electrically stimulated by means of extracellular electrodes. As a result of stimulation Ca²⁺ uptake and release was measured. The uptake response dropped with increasing number of successive stimulation periods and increased with growing stimulus amplitude and duration. Maximum uptake was obtained at 20 V/cm and at least 60 s duration and for temperatures between 10 and 15°C. A Ca²⁺ influx of 700 pmol/1000 cells upon 1 min stimulation was measured at 15°C. This corresponds to an increment of the intraciliary [Ca²⁺] of about $\text{5 · 10}{}^{-4}\text{M}$. Ca²⁺ release was dependent on the stimulus amplitude in a similar manner as was Ca²⁺ uptake. Photographic recordings of the swimming behaviour of the organisms were used to interpret the flux data. At temperatures up to 15°C the cells swam backward perpendicular to the applied electric field of 10 to 20 V/cm. At 25°C they showed forward spiralling movement. For the first time evidence was brought for stimulated Ca²⁺ influx in Paramecium at physiological temperatures. It is concluded from the results that a strong active Ca²⁺ extrusion from the intraciliary space counteracts the influx. The Ca²⁺ pump rate must be at least $\text{8 · 10}{}^{12}$ calcium ions per s per cm² ciliary surface.     

Glycopeptidolipids — a new class of artificial antigens with carbohydrate determinants. Synthesis of artificial antigen with type-specific oligosaccharide hapten fromNeisseria meningitidis group B

Synthetic lipopeptideN-palmitoyltyrosyl-seryl-seryl-asparaginyl-alanine, an analogue of B-mitogenic tripalmitoyl-pentapeptide fromEscherichia coli lipoprotein, was coupled with an oligosaccharide hapten fromNeisseria meningitidis lipooligosaccharide to give a glycopeptidolipid conjugate — the artificial antigen of a new type possessing the type-specific microbial determinant.Abbreviations iBu isobutyl - Bu^t t-butyl - Boc t-butoxycarbonyl - DCC N,N-dicyclohexylcarbodiimide - DMF N,N-dimethylformamide - DMSO dimethylsulfoxide - ONB N-hydroxy-5-norbornen-2,3-dicarboximide ester - ONp 4-nitrophenyl ester - Pal palmitoyl - TEMED N,N,N,N-tetramethylethylenediamine - Z benzyloxycarbonyl - KDO 2-keto-3-deoxyoctonic acid - Hep L-glycero-d-manno-heptose - TPP S-[2,3-bis(palmitoyloxy)-(2RS)propyl]-N-palmitoylcysteinyl-seryl-asparaginyl-alanine.     

Multimeric α-Lactalbumin from Human Milk Induces Apoptosis through a Direct Effect on Cell Nuclei

A fraction from human milk containing spf-multimer α-lactalbumin (MAL) induces apoptosis in tumor cells and immature cells but spares mature cells. The mechanism of apoptosis induction and the molecular basis for the difference in susceptibility between tumor cells and healthy cells have not been defined. In this study we examined the interaction of MAL with different cellular compartments, using confocal microscopy and subcellular fractionation. MAL was shown to accumulate in the nuclei of sensitive cells rather than in the cytosol, the vesicular fraction, or the ER–Golgi complex. Nuclear uptake occurred rapidly in cells that were susceptible to the apoptosis-inducing effect, but not in nuclei of resistant cells. Nuclear uptake was through the nuclear pore complex and was critical for the induction of DNA fragmentation, since inhibition of nuclear uptake with WGA rescued digitonin-permeabilized cells from induction of DNA fragmentation. Ca²⁺was required for MAL-induced DNA fragmentation but nuclear uptake of MAL was independent of Ca²⁺. This way MAL differs from most previously described agents in that it crosses the plasma membrane and cytosol, and enters cell nuclei where it induces DNA fragmentation through a direct effect at the nuclear level.