Mary Edmonds

Polyadenylic Acid Sequences in the Virion RNA of Poliovirus and Eastern Equine Encephalitis Virus

Poliovirus virion RNA contains a single covalently bound sequence of polyadenylic acid which is approximately 49 nucleotides long. A single, slightly longer polyadenylic acid sequence is contained in Eastern Equine Encephalitis virus RNA. Since these viruses are otherwise dissimilar these sequences may play a common role in viral replication, possibly in translation of the viral RNA.

[30] Purification of messenger RNA and heterogeneous nuclear RNA containing poly(A) sequences

Publisher Summary The polyadenylate, poly(A), sequences covalently bound to messenger RNA (mRNA) and to the heterogeneous nuclear RNA (HnRNA) of eukaryotes have served as the basis for their analysis, purification, and isolation on a large scale. The oligodeoxynucleotides, oligo(dt), bound to celluloses have properties that make them particularly suitable for the isolation of the large easily degraded RNA molecules that contain poly(A) sequences. Among these are the covalent nature of the binding and the resistance of polydeoxythymidylate to ribonucleases and alkaline hydrolysis. The fact that the oligo(dT)s attached by methods noted above are relatively short may be particularly advantageous for enhancing the discrimination against weaker interactions involving mismatched or looped-out sequences in these large RNA molecules. This chapter describes the use of oligo(dT) cellulose for the isolation of messenger RNA and heterogeneous nuclear RNA. Some properties of the RNA fractions isolated by these techniques, and the poly(A) sequences derived from them, are presented.

Conversion of radioactive orotic acid into pyrimidine nucleotides of nucleic acid by slices of rat liver.

It has been demonstrated that slices of rat liver have caused the in vitro incorporation of radioactive orotic acid into the pyrimidine nucleotides of nucleic acid and not into the purines. Three milligrams of orotic acid (labeled in the 4 position) were incubated with about 6 g of slices of rat liver for 5 hours at 37°C in a Krebs saline-phosphate buffer of pH 7.4. The solution was saturated with a 95% C02: 5% C02 mixture at the beginning and nothing was used to diminish bacterial action. The slices were homogenized after preliminary washing and the homogenate precipitated with 10% trichloroacetic acid. The extraction of lipids was then carried out with a 3:1 alcohol and ether solution. The nucleic acid was extracted from the protein with hot 10% NaCl and precipitated with alcohol. All of the dried impure nucleic acid (35 mg containing both r.n.a. and d.n.a.) was hydrolyzed with 0.75 cc of N HCl in a boiling water bath for one hour. This hydrolyzed the Purina nucleotides and left the pyramiding nucleotides unchanged. 0.04-0.06 cc were placed on each of 4 filter paper strips 12 cm wide and run with ascending columns of tertiary butyl alcohol (70% made 0.8 N with HCl, 30% water) (1). After drying, the papers were viewed with an ultraviolet Mineralite. The separated bands on paper were eluted with 0.01 N HO, and the solutions read with a Beckman ultraviolet spectrophotometer. The maximum absorptions and the ratios of densities (278-262 mμ for cytidylic and uridvlic acids; 248-262 for guanine and adenine) were determined in order to identify and quantitate the bases. The separate solutions were evaporated on plates and radioactivity determined with a windowless counter. Approximately three-fourths of the hydrolysate from the above experiment containing Purina bases and pyrimidine nucleotides was adjusted to pH 8 and the guanine precipitate removed by centrifugation.

Isolation and characterization of branched oligonucleotides from RNA

Publisher Summary This chapter describes procedures for the isolation, purification, and analysis of the branched oligonucleotides obtained either from RNA labeled in vivo or from intermediates formed in splicing extracts from a specific 32 P-labeled pre-mRNA. Success depends partly on the concentration of branched intermediates in the sample and on the levels of other unidentified components that may cochromatograph with branch points. Branch points, recovered from the digests of RNA lariats purified from nuclear-splicing extracts do not require extensive purification. Secondary analysis is simplified by the removal of the excess mononucleotides generated by nuclease digestion. A DNA template is prepared by inserting—at appropriate restriction sites of a transcriptional cloning vector—a complete intron flanked by exons. The isolation and purification of branched oligonucleotides from mixed RNA populations usually require more elaborate purification schemes, because of the presence in nuclease digests of pre-mRNAs of modified structures, such as caps, methylated nucleotides, and other unidentified components, as well as pNps, which may copurify with branched oligonucleotides because of similarity in net charge.