Impact of RNA isolation protocols on RNA detection by Northern blotting.

We prepared total RNA from the Gram-positive soil bacterium Bacillus subtilis by different RNA extraction procedures to compare their suitability for Northern blot detection of tiny RNAs (~14-mers) or RNAs of intermediate size (100-200 nt) in terms of signal quality, intensity, and reproducibility. Our analysis included two hot phenol methods and two TRIzol extraction procedures. We found that signal intensity/detection sensitivity makes the key difference. Total RNAs prepared by the hot phenol method comprise the length spectrum from tRNAs to large ribosomal RNAs. Larger RNAs are less abundant in TRIzol preparations which instead enrich for RNAs of tRNA size and smaller. Thus, hot phenol methods are the choice for the detection of intermediate-sized and longer RNAs, whereas TRIzol extraction procedures are more suited for the detection of tiny RNAs.

Histidine affinity chromatography of homo-oligonucleotides. Role of multiple interactions on retention.

The recent application of histidine-agarose affinity supports in plasmid purification takes advantage of the biorecognition of nucleic acid bases by the histidine ligand. This consideration prompted the need for better understanding the interactions involved in affinity chromatography of plasmid DNA with the histidine-agarose support. In this work, we used synthetic homo-deoxyoligonucleotides with different sizes (1-30 nucleotides long), to explore the effect of several conditions like hydrophobic character of the individual bases, presence of secondary structures, temperature, pH and salt concentration on the mechanism of retention of nucleic acids to histidine-agarose support. One of the most striking results shows that histidine interacts preferentially with guanine, and the presence of secondary structures on polyA and polyG oligonucleotides has a significant influence on retention. Otherwise, the temperature manipulation has not shown a direct influence on oligonucleotide retention, only inducing conformational changes on secondary structures. Overall, the results obtained provide valuable information for the future development and implementation of histidine and other amino acids as ligands in chromatography for the purification of plasmid DNA and other nucleic acids, by improving the knowledge of the interactions involved as well as of the parameters influencing the retention.

Immunomodulating actions of nucleotides: enhancement of immunoglobulin production by human cord blood lymphocytes.

We have shown previously that polynucleotides enhance in vitro antibody and Ig production in response to T-dependent antigens in mice and augment Ig production by adult human peripheral blood mononuclear cells. Herein, we report their effects on umbilical cord blood mononuclear cells (CBMNC) obtained from full-term babies. CBMNC produced much less IgM/IgG and an almost negligible amount of IgA in response to various stimuli compared with adult peripheral blood mononuclear cells. The supplementation of yeast RNA augmented spontaneous and T-dependent IgM (p < 0.01) but not IgG production by CBMNC. This action was largely attributable to polynucleotides, which appeared to exert their actions in a dose-dependent manner at the initial stages of culture. Their actions were dependent upon the presence of T cells, but they also enhanced spontaneous IgM production by CBMNC in the absence of T cells. Preincubation of T cells from CBMNC and peripheral blood mononuclear cells with RNA for 3 h before the culture resulted in enhanced IgM production, independent of the stimulants used. Thus, polynucleotides appear to exert actions on immature human T cells as well as other lineage cells in vitro. Their actions may be dependent on the presence or absence of antigens or other stimuli and the nature of the stimuli (T dependent versus T independent). These findings may further support the potential importance of nucleotides contained in human breast milk.

Transcription of synthetic DNA containing sequences with dyad symmetry by wheat-germ RNA polymerase II. Increased rates of product release in single-step addition reactions.

Interaction of purified eukaryotic RNA polymerase II with various synthetic palindromic DNA sequences is associated with the formation of transcriptional complexes of different stabilities, i.e. having different propensities for releasing the nascent transcript. This phenomenon was observed by using wheat-germ RNA polymerase II and a series of double-stranded template polymers containing palindromic repeating motifs of 6-16 bp, with regulatory alternating purine and pyrimidine bases such as d[ATA(CG)nC].d[TAT(GC)nG], with n = 1, 3 or 6 referred to as d(GC), d(GC)3 or d(GC)6, respectively. We also synthesized two double-stranded methylated polymers, containing the repeating units d(ATAm5CGm5C).d(TATGm5CG) and d[ATA(m5CG)6m5C].d[TAT(Gm5C)6G] [designated d(GmC) and d(GmC)6, respectively]. All of these polymers served as templates for the reaction of single-step addition of CTP to a CpG primer catalysed by wheat-germ RNA polymerase II, to an extent that seems well correlated with the number of potential initiation sites within the DNA molecules. Furthermore, in these reactions, the enzyme appears to form relatively stable transcriptional complexes, as trinucleotide product was released only very slowly. In marked contrast to the results with the CpG primer, the single-step addition reaction primed by UpA, i.e. the synthesis of UpApU proceeded at a much higher velocity and was strongly enhanced by increasing the d(G-C) content of the repeating units of the DNA polymers. Thus, taking into account the number of potential sites at which UpApU synthesis could occur, the extent of UpApU synthesis was increased about 12-fold with d(GC)6 compared to that with the d(GC) template. The catalytic nature of the reaction necessarily implies that the stability of the transcription complexes with the plant RNA polymerase II decreased as the d(G-C) content of the repeating motif increased. Furthermore, although the synthesis of CpGpC could be demonstrated with d(GmC)6 as template, the UpA-primed synthesis of UpApU could not be detected with this polymer. The results obtained in transcription of these polymers are discussed in relation to the potential involvement of palindromic DNA in transcription termination and attenuation in the presence of RNA polymerase II.

Analysis of the secondary structure of the poly(C) tract in foot-and-mouth disease virus RNAs.

Sodium bisulphite modification of foot-and-mouth disease virus (FMDV) RNA in solution indicates that the majority of the poly(C) tract in the RNA is single-stranded in concordance with previous results with encephalomyocarditis virus RNA. The reaction kinetics are biphasic; 60% of the cytidylic acid in the poly(C) tract reacts like synthetic poly(C), and the remainder with the kinetics of the cytidylic acid in the rest of the RNA. The reactivity of the poly(C) tract with poly(I) indicates that it is looped out and exposed in the RNA. The deamination reaction has also been used to investigate the structure of the replicative form (RF) and replicative intermediate (RI) isolated from infected cells. Analysis by gel electrophoresis of the long RNase A- and T1-resistant oligonucleotides of RI suggests that it has five single-stranded poly(C) tracts to every one which is base-paired. Bisulphite reactivity of the poly(C) tract and gel electrophoresis of the ribonuclease-resistant oligonucleotides of RF indicate that the poly(C) is base-paired to a poly(G) tract in this molecule. The presence of a poly(G) tract in RF and RI provides unequivocal evidence that the poly(C) is replicated via poly(G) in the negative strand.

Hydrophobic affinity chromatography of nucleic acids and proteins. II. Activity of trityl Sepharose immobilized enzymes.

A variety of nucleic acid synthetic and degradative enzymes and proteases are shown to bind to trityl sepharose columns and, for the most part, retain moderate amounts of activity for periods of days to weeks. Non-covalent hydrophobic interactions are believed to be largely responsible for the observed binding and maintenance of activity. In addition the hydrophobic binding mechanism of poly A to trityl sepharose columns under a variety of conditions is compared with that to nitrocellulose columns and contrasted with that of dT cellulose columns.

Poly(U)-agarose affinity chromatography: specific, sensitivity selectivity, and affinity of binding.

These studies were done to determine four basic intrinsic properties of poly(U)-agarose affinity columns. Specificity of binding studies demonstrated that binding to these columns is highly specific with greater than 90% complementary binding and less than or equal to 3% noncomplementary binding. Sensitivity of binding studies indicated that a minimum sequence of 10 adenylates is required for detectable complementary binding. Selectivity of binding studies revealed that nonsequential adenylates in native RNAs and randomly distributed adenylates in synthetic poly(A)-poly(C) co-polymers did not bind to poly(U)-agarose affinity columns. Whereas, affinity of binding studies demonstrated that A=U complementary base pairing is independent of chain-lengths of greater than or equal to 25 adenylates and dependent of chain-lengths of less than 25 adenylates. Thus the data demonstrates that poly(U)-agarose affinity chromatography is scientifically sound and expedient for the detection and isolation of poly(A)-containing cellular and viral RNAs.

[Molecular weight distribution of hydrolysis products of polyuridylic acid by cobra venom ribonuclease. Preparation of polynucleotides of required molecular weight]. / Izuchenie raspredeleniia po molekuliarnym vesam produktov gidroliza poliuridilovoi kisloty ribonukleazoi iz iada kobry. Preparativnoe poluchenie polinukleotidov s zadannym molekuliarnym vesom.

The composition of polyuridilic acid hydrolisis products with mol. weight 400.000--600.000 by endoribonuclease from cobra venom was studied. The molecular weight distribution of products formed at initial stages of hydrolysis was according to the regularities of accidental cleavage of phosphodiether linkages. The sufficient non-statistic regularities due to the high-molecular polynucleotide hydrolysis for the most part during further hydrolysis were observed out of the way of accumulation of oligonucleotides with a polymerity degree of about 15--20. As shown from the oligocytidilates hydrolysis, the velocity of process increased sufficiently with increasing substrate polymerity degree. The results obtained were used for preparation of polyuridilic fractions with a narrow range of molecular weight distribution.