Interaction of 6-thiopurines and thiol-containing RNA with a cellulose mercurial.

Thiol-containing bases such as 6-mercaptopurine bind to mercurated cellulose under conditions where non-thiol bases do not bind. Both RNA and thiol-containing RNA bind to mercurated cellulose, but the affinity for thiol-containing RNA is considerably greater. Ligands such as CN^? and $\text{S}{}_2\text{O}{}_3{}^{\mathrm{2?}}$ inhibit this binding to mercurated cellulose, and conditions can be found which permit almost complete retention, of thiol-containing RNA with low retention of non-thiol-containing RNA. A simple method is described for separating newly synthesised RNA containing thiol groups from older RNA which does not contain thiol groups.     

Sulfhydrylcellulose: a new medium for chromatography of mercurated polynucleotides

We have synthesized a new medium, sulfhydrylcellulose, for affinity chromatography of mercurated polynucleotides. It is the product of reaction between aminoethylcellulose and N-acetylhomocysteine thiolactone. Sulfhydrylcellulose carries up to 90 mumol of SH groups/g and is inexpensive, easy to prepare, and stable. Because it binds mercurated RNA specifically and reversibly and exhibits no size discrimination, sulfhydrylcellulose should have wide applications.     

New procedure for isolation of Rous sarcoma virus-specific RNA from infected cells.

The use of mercurated "strong stop" complementary DNA (complementary to the 5'-terminal 101 nucleotides of Rous sarcoma virus RNA) in the isolation of virus-specific RNA from infected chicken embryo fibroblasts is described. Strong stop Rous sarcoma virus complementary DNA was mercurated chemically, and, as a result of the low complexity of this DNA, short hybridization times (up to 15 min) and heating in the absence of formamide were found to be adequate conditions for the isolation of virus-specific RNA. The purity of the isolated RNA was demonstrated by analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. The isolated RNA could be translated in the in vitro protein synthesis system derived from rabbit reticulocytes, and an analysis of polypeptides programmed by isolated RNA before and after immunoprecipitation further demonstrated both the purity of the isolated mRNA and the quantitative nature of the isolation procedure.     

A new hybridocytochemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands. II. Effects of variations in ligand structure on the in situ detection of mercurated probes

In the preceding paper, a method to detect specific DNA sequences with mercurated nucleic acid probes and sulfhydryl-hapten ligands has been described. Due to the instability of the bond between mercury and a negatively charged sulfhydryl-hapten ligand (trinitrophenyl-glutathione), the in situ formed hybrid could not be detected. On basis of model system experiments it was suggested that this mercury-sulfhydryl bond could be stabilized by an extra polar interaction between ligand and nucleic acid. This was achieved by reversing the net charge of the ligand. Such ligands were synthesized by reacting aliphatic diamines to the carboxyl groups of Tnp-glutathione using a water soluble carbodiimide. Gel chromatographic analysis of mercurated polynucleotide-ligand complexes showed that the stability of the mercury-sulfhydryl bond is increased by the reversal of the net charge of the ligand. In situ hybridized mercurated mouse satellite DNA to mouse liver nuclei and mercurated kinetoplast cRNA hybridized to Crithidia fasciculata were immunocytochemically detected after the introduction of these positively charged ligands. The described method is applicable for RNA and DNA probes. It has a sensitivity comparable to other non-autoradiographic methods, is relatively simple to perform and can be carried out with ordinary laboratory chemicals.     

A new hybridocytochemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands

Summary In the preceding paper, a method to detect specific DNA sequences with mercurated nucleic acid probes and sulfhydryl-hapten ligands has been described. Due to the instability of the bond between mercury and a negatively charged sulfhydryl-hapten ligand (trinitrophenyl-glutathione), the in situ formed hybrid could not be detected. On basis of model system experiments it was suggested that this mercury-sulfhydryl bond could be stabilized by an extra polar interaction between ligand and nucleic acid. This was achreved by reversing the net charge of the ligand. Such ligands were synthesized by reacting aliphatic diamines to the carboxyl groups of Tnp-glutathione using a water soluble carbodiimide. Gel chromatographic analysis of mercurated polynucleotide-ligand complexes showed that the stability of the mercury-sulfhydryl bond is increased by the reversal of the net charge of the ligand.In situ hybridized mercurated mouse satellite DNA to mouse liver nuclei and mercurated kinetoplast cRNA hybridized to Crithidia fasciculata were immunocytochemically detected after the introduction of these positively charged ligands.The described method is applicable for RNA and DNA probes. It has a sensitivity comparable to other non-autoradiographic methods, is relatively simple to perform and can be carried out with ordinary laboratory chemicals.This investigation was supported by the Netherlands Foundation for Medical Research Fungo (grant nr 13-54-21)     

Selective isolation of mercurated DNA by affinity chromatography on thiol matrices

A method for isolating picomole quantities of nascent mercurated DNA from a mixture of cellular nucleic acids using affinity chromatography on thiol-agarose is described. Analysis of mercurated DNA (HgDNA) isolated in the presence of in vivo-labeled cellular RNA or in vitro-synthesized RNA showed a low level of RNA contamination, about 0.04-0.16%, in the HgDNA. Comparative binding studies on different thiol matrices showed that the efficiency of binding of HgDNA was related to the nature but not to the SH content of the matrix used. Another important parameter for binding was the structure of HgDNA. The recovery was 98% with large nascent HgDNA sedimenting at about 30 S, whereas for short pulse-labeled single-stranded HgDNA (20-50 nucleotides long), the maximum recovery was 60%. The effect of the structure of HgDNA on the binding to the thiol matrix was probed using a variety of well-defined mercurated structures obtained from phage DNA and their restriction fragments. For DNA containing one 5-mercuricytidine 5'-triphosphate (HgdCMP) residue at each 3'-end, short fragments (size range, 230-510 bp) were bound quantitatively. With larger fragments (size range, 490-1100 bp), the binding decreased progressively with increasing size. DNA fragments larger than 1060 bp did not bind to the matrix. Single-stranded DNA containing only one HgdCMP at one end did not bind to the matrix even in the size range 200-1100 nucleotides. In contrast, continuous stretches of HgdCMP residues in one strand or short stretches of HgdCMP residues at random in both strands permit quantitative binding irrespective of size.     

A method for the isolation of segments from the 5′ ends of retrovirus RNA

A method for the isolation of segments of any desired length from the 5′ end of retrovirus RNA has been tested. The method is based on selection of 5′-specific segments by hybridizing suitably fragmented genomic (35 S) RNA to mercurated strong stop cDNA followed by chromatography on sulfhydryl-agarose. The method has been shown to be effective for Akv viral RNA by observing the T1 oligonucleotide fingerprints of a 5′-enriched fraction. This fingerprint pattern is of lower complexity than that of total 35 S RNA, contains oligonucleotide spots that have previously been assigned as 5′ specific by conventional fingerprinting methods, and does not overlap with the pattern from 3′-specific RNA.     

Direct covalent mercuration of nucleotides and polynucleotides.

Nucleotides of cytosine and uracil are readily mercurated by heating at 37-50 degrees in buffered aqueous solutions (pH 5.0-8.0) containing mercuric acetate. Proton magnetic resonance, elemental, electrophoretic, and chromatographic analyses have shown the products to be 5-mercuricytosine and 5-mercuriuracil derivatives, where the mercury atom is covalently bonded. Polynucleotides can be mercurated under similar conditions. Cytosine and uracil bases are modified in RNA while only cytosine residues in DNA are substituted. There is little, if any, reaction with adenine, thymine, or guanine bases. The rate of polymer mercuration is, unlike that of mononucleotides, markedly influenced by the ionic strength of the reaction mixture: the lower the ionic strength the faster the reaction rate. Pyrimidine residues in single- and double-stranded polymers react at essentially the same rate. Although most polynucleotides can be extensively mercurated at pH 7.0 in sodium or Trisacetate buffers, tRNA undergoes only limited substitution in Tris buffers. The mild reaction conditions give minimal single-strand breakage and, unlike direct iodination procedures, do not produce pyrimidine hydrates. Mercurated polynucleotides can be exploited in a variety of ways, particularly by crystallographic and electron microscopic techniques, as tools for studying polynucleotide structure.     

Isolation of Rhizobium loti Strain-Specific DNA Sequences by Subtraction Hybridization

Mixed-phase (heterogeneous) and single-phase (homogeneous) DNA subtraction-hybridization methods were used to isolate specific DNA probes for closely related Rhizobium loti strains. In the heterogeneous method, DNA from the prospective probe strain was repeatedly hybridized to a mixture of DNA from cross-hybridizing strains (subtracter DNA) which was immobilized on an epoxy-activated cellulose matrix. Probe strain sequences which shared homology with the matrix-bound subtracter DNA hybridized to it, leaving unique probe strain sequences in the mobile phase. In the homogeneous method, probe strain sequences were hybridized in solution to biotinylated, mercurated subtracter DNA. Biotinylated, mercurated subtracer DNA and probe strain sequences hybridized to it were removed by two-step affinity chromatography on streptavidin-agarose and thiol-Sepharose. The specificity of the sequences remaining after subtraction hybridization by both methods was assessed and compared by colony hybridization with R. loti strains. Both methods allowed the rapid isolation of strain-specific DNA fragments which were suitable for use as probes.