Analysis of double-stranded poly(A).poly(U) molecules by reversed-phase high-performance liquid chromatography.

The behaviour of different batches of synthetic Poly(A).Poly(U) in reversed-phase high-performance liquid chromatography (HPLC) was studied. They consist of large molecules mainly in the form of a double strand. Differences in the elution patterns were correlated with properties detected by conventional methods such as electrophoresis, centrifugation, fusion analysis or enzymatic digestions. Under the present conditions, contamination by products and precursors used during synthesis was detectable, but was absent in most of the preparations. The differences in elution patterns between batches appear to be correlated with the size of the molecules synthesized. The chromatograms suggested that Poly(A).Poly(U) molecules contain single-strand portions at least transiently. The presence of such portions was confirmed by enzymatic digestion with S1 nuclease. The rapidity, reproducibility and ease of reversed-phase HPLC qualify this technique as a tool for routine analysis.

[Interaction of natural and synthetic polynucleotides with liposomes in the presence of divalent cations]. / Vzaimodeistvie prirodnykh i sinteticheskikh polinukleotidov s liposomami v prisutstvii dvukhvalentnykh kationov.

The formation of complexes of polynucleotides (DNA, poly A.poly U) with liposomes from egg lecithins, L-alpha-phosphatidylcholine, dimirystoyl and other lipids in the presence of divalent cations was studied by differential scanning microcalorimetry circular dichroism and turbidimetry. It was shown that the secondary structure of polynucleotides (double or triple helix) was necessary for the formation of these complexes. This structure was partially destroyed during formation of complexes. It was shown, that three main types of lipids, i.e. phosphatidylcholine, phosphatidylethanolamine and sphingomyelin participate in interactions between liposomes, polynucleotides and Mg2+.

[1-(3-C-methyl-beta-D-ribofuranosyl)uracil-5'-phosphate--a terminator of RNA synthesis catalyzed by RNA polymerase from E. coli]. / 1-(3'-C-metil-beta-D-ribofuranozil)uratsil-5'-trifosfat--terminator sinteza RNK, kataliziruemogo RNK-polimerazoi E. coli.

It is shown that 1-(3'-C-methyl-beta-D-ribofuranosyl)uracil 5'-triphosphate is a terminator of RNA synthesis and may be used for nucleic acid sequencing with DNA-dependent RNA polymerase from E. coli.

RNA-like conformational properties of a synthetic DNA poly(dA-dU).poly(dA-dU).

Differences in the circular dichroism of poly(dA-dT).poly(dA-dT) and poly(dA-dU).poly(dA-dU) and in its temperature induced changes are reported. A comparison to the data obtained with DNA and RNA indicates that an absence of thymine methyl groups in the polynucleotide results in promoting its RNA-like conformational properties. However, poly(dA-dU).poly(dA-dU) is not an A-DNA type of double helix.

Cytochemical hybridization with fluorochrome-labeled RNA. I. Development of a method using nucleic acids bound to agarose beads as a model.

A new procedure to label RNA at the 3'-terminus with a fluorochrome molecule is described. The thiosemicarbazides derived from tetramethylrhodamine isothiocyanate (TRITC) and fluorescein isothiocyanate (FITC) were prepared by reacting these compounds with hydrazine in dimethylsulfoxide (DMSO):pyridine, 99:1 (v/v). They coupled efficiently to the aldehydes generated by periodate oxidation of RNAs. We determined, using Sepharose to which different nucleic acids and proteins had been bound, that the label added no specific binding properties to the RNA, and did not interfere with duplex formation of labeled poly(U) and poly(A). The stability of the fluorochrome-RNA bond under conditions generally used for hybridization was investigated. The bond was found to be unstable at 66 degrees C in 3 x SSC, 0.1% SDS (50% loss within 45 min) but stable for at least 40 hr at 23 degrees C in 70% formamide/3 x SSC. The hybridization characteristics of complementary RNA, both fluorochrome- and 3H-labeled, were investigated using DNA-Sepharose beads as a cytochemical model. Hybridization was measured by scintillation counting of microliter quantities of beads and quantitative fluorescence microscopy of individual Sepharose beads. No influence of the label on the specificity and stability of the hybrids was found. Maximum specific fluorescence was found after hybridization at 23 degrees C in 70% formamide/3 x SSC. These results made possible the successful use of fluorochrome-labeled RNA to perform cytochemical hybridization followed by detection of the hybrids with fluorescence microscopy. This will be described in an accompanying article.

A circular dichroism study of Escherichia coli Initiation Factor-1 binding to polynucleotides.

Binding of Escherichia coli Initiation Factor-1 protein to the nucleic acid lattice induces alterations in the secondary structures of a variety of purine and pyrimidine containing polynucleotides in both the single and double stranded conformations, as assessed by circular dichroism spectroscopy. The helical hairpin form of poly(U), the single-stranded stacked form of poly(C), and the duplex poly(A) x poly(U) (in the presence of Mg2+) are stoichiometrically converted by Initiation Factor-1 (IF-1) to structures spectrally indistinguishable from their partially or completely thermally denatured forms. By contrast, the binding of IF-1 to double stranded poly(C), single- and double-stranded poly(A) elicited spectral responses which were interpreted in terms of diminished base-base interaction, not equivalent to that induced by thermal means. Stoichiometric endpoints of 3-5 nucleotide residues/IF-1 were determined for polynucleotide structures in those cases where light scattering artifacts at low nucleotide residue to protein ratios were absent. In the absence of Mg2+ IF-1 was unable to elicit a conformation alteration effect in poly(A) x poly(U), while for poly(U) much less of an effect was observed than in the presence of this divalent ion. The functional significance of these results is briefly considered.

Immunochemical characterization of the anti-RNA antibodies found in scleroderma and systemic lupus erythematosus. I. Differences in reactivity with Poly (U) and Poly-(A) Poly (U).

In a previous study, all 40 sera from patients with scleroderma, 20 of 40 sera from SLE patients, but none of 40 sera from normal controls, were found to have antibodies to ssRNA. All scleroderma sera were also found to react with HSA-coupled uridine and UMP and their reaction with HSA-coupled uridine and UMP and their reaction with ssRNA could be inhibited by uracil, uridine, and UMP. To characterize further these uracil-specific anti-RNA antibodies found in scleroderma and compare them with the anti-RNA antibodies found in SLE, we tested their reactivity with Poly (U) and with Poly (A)-Poly (U) and all but one failed to react with Poly (A)-Poly (U). This same serum was the only one in which the reaction with Poly (U) could not be inhibited with uracil. Reactivity of SLE sera was strikingly different from that found in scleroderma sera. Seventeen of 34 SLE sera studied reacted with ssRNA but only four of these reacted with Poly (U). Conversely, two SLE sera that reacted with Poly (U) did not react with ssRNA. Fifteen reacted with Poly (A)-Poly (U) and only two of these failed to react with ssRNA. Five SLE sera which were reactive with ssRNA did not precipitate with Poly (A)-Poly (U). All SLE sera which reacted with Poly (U) could be inhibited with uracil, although less effectively than in scleroderma. Reactivity with Poly (A)-Poly )U) was not inhibited with uracil nor with adenosine. These findings confirm that antibodies to RNA that are found in scleroderma are directed to uracil and thus specific to ssRNA, whereas RNA antibodies found in SLE sera are heterogeneous and directed to either the base, to the site of union of the base and sugar moiety to the ribose backbone, or to the helical structure of double stranded RNA. These differences and the respective antigenic specificities of these anti-RNA antibodies found in scleroderma and SLE may be theoretically important.