The accessibility of phage MS2 RNA to structure specific nucleases in various conditions

The accessibility of ds-and ss-segments of phage MS2 RNA to ds-and ss-specific nucleases (RNase III, nuclease SV and nuclease S1) was studied. The results show that the RNA has hydrolysis sites for all the nucleases used. These sites are unvariable in a wide range of the conditions (ionic strength, pH, bivalent cations and temperature) and are not changed also after denaturation-renaturation of the RNA. This testifies that the distribution and interactions of ds-and ss-segments in the whole molecule are very specific and stable.     

Aliphatic analogues of nucleotides: synthesis and affinity towards nucleases

DL-1-(2,3-Dihydroxypropyl)thymine was prepared by Hilbert-Johnson reaction of 2,4-dinethoxy-5-methylpyrimidine with allyl bromide followed by the osmium tetroxide catalyzed hydroxylation of the l-allyl-4-methoxy-5-methylpyrimidin-2-one obtained as an intermediate. The D-glycero enantiomer, R-1-(2,3-dihydroxypropyl)thymine and the corresponding 1-substituted uracil derivative were prepared from 3-O-p-toluenesulfonyl-1, 2-O-isopropylidene-D-glycerine and sodium salt of 4-methoxy-5-methylpyrimidin-2-one or 4-methoxypyrimidin-2-one followed by treatment with hydrogen chloride in ethanol. The phosphorylation of the above 2,3-dihydroxypropyl derivatives with phosphoryl chloride in triethyl phosphate afforded the corresponding 3-phosphates which were transformed into the 2′,3′-cyclic phosphates by the condensation with N,N′-dicyclohexylcarbodiimide. The latter compounds of the D-glycero configuration are split by some microbial RNases to the 3-phosphates.     

NADP-Agarose: an affinity adsorbent for tobacco extracellular nuclease and other nucleases.

A general method for the isolation of modified tryptophan-containing peptides is described, which takes advantage of the adsorption properties of tale for aromatic nitrocompounds. The method has been extensively assayed on tryptic and chymotryptic hydrolyzates of human Bence Jones proteins of k and λ type and of human serum albumin, and in preliminary experiments on egg white lysozyme and rabbit IgG light chains. All those proteins had been previously carboxymethylated and modified with 2,4 dinitrophenylsulfenylchloride, a selective reagent for tryptophan.     

Derivatization of epoxy-activated agarose with various carbohydrates for the preparation of stable and high-capacity affinity adsorbents: Their use for affinity chromatography of carbohydrate-binding proteins

Two types of affinity adsorbents for lectins were prepared by new simple procedures. Both types of adsorbents had high ligand concentration and chemically stable linkage between ligand and Sepharose 4B. Oligosaccharide ligands were coupled by reductive amination with sodium cyanoborohydride to amino-Sepharose 4B prepared by amination of epoxy-activated Sepharose 4B. The glycamyl-Sepharose 4B thus obtained had particularly high adsorption capacities for lectins; lactamyl-Sepharose 4B, 58 mg/l ml of gel for peanut lectin; maltamyl-Sepharose 4B, 146 mg/ml for concanavalin A; and tetra-N-acetylchitotetraamyl-Sepharose 4B, 36 mg/ml for wheat germ agglutinin. Hexosamine was coupled by the aid of carbodiimide to carboxyl-Sepharose 4B prepared by succinylation of amino-Sepharose 4B. Galactosamine-Sepharose 4B adsorbed 145 mg soybean agglutinin/l ml gel. The columns turned from a semitransparent white to a milky white as they were saturated with lectins.     

Streptomyces flavogriseus cellulase: Evaluation under various hydrolysis conditions

Streptomyces flavogriseus CMCase and Avicelase were very stable at 30 degrees C but not at 40 degrees C or higher. beta-Glucosidase was less stable at all temperatures tested. Stabilities were similar at pH values between 5.5 and 7, the optimal range for enzyme activity. Cellulose solubilizing activity was reduced by 40% at a cellobiose concentration of 150mM but glucose inhibited activity by only 10% at this concentration. beta-Glucosidase was inhibited by 40% at a glucose concentration of 10mM (ten times the substrate concentration). Relatively dilute S. flavogriseus cellulase extensively hydrolysed acid-swollen cellulose at concentrations as high as 10%. More highly crystalline forms of cellulose were more resistant to attack.     

The ARiBo tag: a reliable tool for affinity purification of RNAs under native conditions

Although RNA-based biological processes and therapeutics have gained increasing interest, purification of in vitro transcribed RNA generally relies on gel-based methods that are time-consuming, tedious and denature the RNA. Here, we present a reliable procedure for affinity batch purification of RNA, which exploits the high-affinity interaction between the boxB RNA and the N-peptide from bacteriophage λ. The RNA of interest is synthesized with an ARiBo tag, which consists of an activatable ribozyme (the glmS ribozyme) and the λBoxB RNA. This ARiBo-fusion RNA is initially captured on Glutathione-Sepharose resin via a GST/λN-fusion protein, and the RNA of interest is subsequently eluted by ribozyme self-cleavage using glucosamine-6-phosphate. Several GST/λN-fusion proteins and ARiBo tags were tested to optimize RNA yield and purity. The optimized procedure enables one to quickly obtain (3 h) highly pure RNA (>99%) under native conditions and with yields comparable to standard denaturing gel-based protocols. It is widely applicable to a variety of RNAs, including riboswitches, ribozymes and microRNAs. In addition, it can be easily adapted to a wide range of applications that require RNA purification and/or immobilization, including isolation of RNA-associated complexes from living cells and high-throughput applications.     

Aptamer affinity chromatography:: combinatorial chemistry applied to protein purification

The systematic evolution of ligands by exponential enrichment process is a combinatorial chemistry method that allows the identification of specific oligonucleotide sequences, known as aptamers, that bind to a desired target molecule with high affinity and specificity. Here, a DNA-aptamer specific for human -selectin was immobilized to a chromatography support to create an affinity column. This column was effectively applied as either the first or second step in the purification of a recombinant human -selectin–Ig fusion protein from Chinese hamster ovary cell-conditioned medium. The fusion protein was efficiently bound to the column and efficiently eluted by gentle elution schemes. Application of the aptamer column as the initial purification step resulted in a 1500-fold purification with an 83% single step recovery. These results demonstrate that oligonucleotide aptamers can be effective affinity purification reagents.     

Hydrophilic-hydrophobic properties of microorganisms under various culturing conditions

The index of hydrophobicity and the hydrophilic-hydrophobic properties of seven microbial cultures were determined by studying their adhesion to n-hexadecane. The index of hydrophobicity was shown to be a stable characteristic of a culture grown in media with different carbon sources. As was found for Escherichia coli K-12 and Acinetobacter calcoaceticus K-9 whose hydrophobicity indicates were quite different, the character of cell hydration was virtually independent of the growth phase and did not change upon either submerged or superficial cultivation.     

Variability of Bacillus thuringiensis under various growth conditions

When a lysogenic culture of Bacillus thuringiensis subsp. galleriae 69-6 was grown under the batch conditions, 93-99% of cells in the population produced R-form colonies and ca. 1% yielded S-form colonies. The amount of spore-forming cells was 99% in R-variants and 8% in S-variants. The quantity of S-variants rose abruptly to 99% when the culture was grown under the chemostat conditions. The number of S-variants increased with the rate and the duration of growth. The process was influenced by growth-limiting factors. Temperate phage variants capable of host culture lysis on solid media (i.e. h-mutants) were not found under the conditions of batch cultivation. However, such phage particles (h-mutants) appeared under the conditions of chemostat. The titre of these phage particles reached 10(8), 10(7) and 10(4) particles per 1 ml at limitation with yeast extract, glucose and phosphorus, respectively. Under the conditions of chemostat, the particles behaved as temperate ones and their growth was not found. Irrespective of the limitation, the phage titre did not correlate with the ratio of R and S-forms in the population. When the growth was limited with phosphorus, the quantity of S-forms increased abruptly while the spontaneous induction of the phage was inhibited. The quantity of cells capable of spore formation decreased in the cultures isolated from the chemostat and grown on MPA: 69-80% of the cells in R-forms and merely 8% in S-forms.     

Methacryloylamidohistidine in affinity ligands for immobilized metal-ion affinity chromatography of ferritin

A new metal-chelate adsorbent utilizing 2-methacryloylamidohistidine (MAH) was prepared as a metalchelating ligand. MAH was synthesized using methacryloly chloride and histidine. Monosize nanospheres with an average diameter of 450 nm were produced by emulsion polymerization of 2-hydroxyetylmethacrylate (HEMA) and MAH. Then, Fe³⁺ ions were chelated directly onto the monosize nanospheres. Mon-poly(HEMA-MAH) nanospheres were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and elemental analysis. Fe³⁺ chelated monosize nanospheres were used in ferritin adsorption from an aqueous solution. The maximum ferritin adsorption capacity of Fe³⁺-chelated mon-poly(HEMAMAH) nanospheres was 202 mg/g at pH 4.0 in acetate buffer. The non-specific ferritin adsorption on the monpoly( HEMA-MAH) nanospheres was 20 mg/g. The adsorption behavior of ferritin could be modeled using both Langmuir and Freundlich isotherms. The adsorption capacity decreased with increasing ionic strength of the binding buffer. High desorption ratios (> 95% of the adsorbed ferritin) were achieved with 1.0 M NaCl at pH 7.0. Ferritin could be repeatedly adsorbed and desorbed with the Fe³⁺-chelated mon-poly(HEMA-MAH) nanospheres without significant loss of adsorption capacity.