Optimization of the efficiency of cross-linking PtII oligonucleotide phosphorothioate complexes to complementary oligonucleotides.

We have investigated the efficiency with which PtII complexes cross-link phosphorothioates of oligonucleotides to complementary DNA targets. The A and G residues 2-5 bases downstream from the 5'-phosphorothioate group are preferred sites for cross-linking. Replacement of residues in this part of the target by T residues results in greatly decreased cross-linking when cis platinum diammine dichloride (cisPtII) or potassium platinous chloride (K2PtCl4) are used. Trans platinum diammine dichloride (transPtII) forms cross-links with T residues if A and G residues are absent from the susceptible region of the target. Oligomers containing an internal phosphorothioate group can also be linked to their templates with transPtII, but not with cisPtII or K2PtCl4. Cross-linking via an internal phosphorothioate group tends to be less efficient than cross-linking via a 5'-terminal phosphorothioate. The Sp isomers of internal phosphorothioates are cross-linked more efficiently than the Rp isomers. Preliminary experiments suggest that the efficiency of cross-linking to RNA targets will prove similar to that found for DNA targets.     

A simple assay procedure for beta-D-mannanase.

A simple assay procedure for beta-D-mannanase enzyme has been developed which employs carob D-galacto-D-mannan dyed with Remazolbrilliant Blue. Additionally, the procedure is quantitative, relatively sensitive, and highly specific for beta-D-mannanase enzyme. It can be readily used for the determination of beta-D-mannanase activity in crude enzyme preparations and column-chromatography eluates.     

A simple procedure for tenascin purification.

Here we describe a two-step procedure for purification of human tenascin from conditioned medium of the SK-MEL-28 human melanoma cell line. The first step consists in passing the conditioned media through two chromatography columns connected in sequence. The first is a large capacity gelatin--Sepharose affinity chromatography column (to remove fibronectin), the second, over which the unbound material from the first column flows directly, is a hydroxyapatite chromatography column. Under these conditions, all tenascin present in the conditioned medium binds to the hydroxyapatite chromatography column from which it is then eluted by a 5-300 mM sodium phosphate gradient. With this step, we obtain a crude tenascin preparation, concentrated about 20 times with respect to the starting conditioned medium, and in which tenascin represents more than 50% of the total protein. The second step consists of two sequential precipitations with 6% and 12.8% poly(ethylene glycol). After this step, tenascin is more than 95% pure and does not show any contamination of chondroitin-sulfate-containing proteoglycans that are known to bind to it. From 21 medium we obtain about 3-4 mg tenascin which corresponds to a yield of about 40-50%. This procedure gives a higher yield, is simpler with respect to procedures previously described, avoids the exposure of the protein to denaturing agents or harsh conditions and could be used for purification of tenascin from the conditioned media of other cell lines. Thus, this procedure may represent a simple and useful tool for the preparation of tenascin to study its biological functions.     

A simple purification procedure for monomeric nucleosomes.

The soluble chromatin fragments from nuclei of avian erythrocytes digested with micrococcal nuclease were fractionated by the addition of sodium phosphate to 0.1 m. The supernatant consisted predominantly of monomeric nucleosomes, while most dimeric and larger nucleosomes precipitated. A variable percentage of monomers also precipitated, the exact amount depending upon the extent of digestion. The solubility properties can be used for the simple preparation of fractions that are highly enriched for monomers either containing, or deplete in, lysine-rich histone.     

A simple procedure for identification ofClostridium botulinum colonies

For direct identification of toxigenic colonies ofClostridium botulinum type E, suspected colonies are uniformly suspended in a phosphate buffer containing 0.5% (w/v) gelatin and 0.05% (w/v) Tween 20. After centrifuging, the supernatant is tested for botulinal toxin by an enzyme-linked immunosorbent assay (ELISA). The assay is specific for this type as it did not react with culture filtrates of otherClostridium species, including non-toxigenic E-like organisms.     

A simple procedure for preparing substrate for Pleurotus ostreatus cultivation

The use of wooden crates for composting a mixture of 70% grass, (Digitaria decumbens), and 30% coffee pulp, combined with 2% Ca(OH)(2), was studied as a method for preparing substrate for the cultivation of Pleurotus ostreatus. Crate composting considerably modified the temperature pattern of the substrate in process, as compared to pile composting, where lower temperatures and less homogeneous distributions were observed. Biological efficiencies varied between 59.79% and 93% in the two harvests. Based on statistical analysis significant differences were observed between the treatments, composting times and in the interactions between these two factors. We concluded that it is possible to produce P. ostreatus on a lignocellulosic, non-composted, non-pasteurized substrate with an initial pH of 8.7, and that composting for two to three days improves the biological efficiency.     

A simple procedure for the purification of rat alpha-fetoprotein

A simple purification procedure for obtaining a high yield of electrophoretically and immunologically pure rat α-fetoprotein from amniotic fluid is described. Rat amniotic fluid is passed through an anti-rat albumin immunoabsorbent column to remove albumin. The albumin-free eluate is then chromatographed on DEAE-Sephacel to separate α-fetoprotein from transferrin and other minor protein contaminants. This two-step purification procedure results in a recovery of approximately 70% of the rat α-fetoprotein originally present in the amniotic fluid.     

A simple procedure for large-scale purification of plasmid DNA

We report a simple, rapid and reliable procedure for large-scale purification of plasmid DNA from non-amplified bacterial cultures. It is a modification of the boiling method of Holmes and Quigley [Anal. Biochem. 114 (1981) 193-197] and involves gel-filtration chromatography using Sephacryl S-1000 for final purification of plasmid DNA. This method does not require CsCl gradients and the recovered plasmids are free of RNA and chromosomal DNA, are supercoiled, retain their biological activity, and are suitable for restriction analysis.     

A simple procedure for isolating adenosine triphosphatase from mitochondria

A simple method for isolation of adenosine triphosphatase (EC 3.6.1.3) from mitochondria is described. The enzyme is released from mitochondrial Lubrol particles by drastic sonication and purified by gel filtration on Sepharose 6-B. The described procedure is effective in isolating adenosine triphosphatase from rat liver as it is from beef heart mitochondria. The enzyme isolated from beef heart has a specific activity of 120 μmol P/min per mg protein and enzyme isolated from rat liver has a specific activity of 70 μmol P/min per mg protein when measured as a release of inorganic phosphate.