Distribution patterns for 5-methylcytosine among apurinic DNAs from several sources

Abstract

Purified DNA from the liver of rats, mice, rabbits, and guinea pigs, from guinea pig lymph nodes, from hyperplastic nodules induced in rat liver by feeding with 2-(acetylamino)fluorene, and from Escherichia coli cells was made apurinic by reaction with diphenylamine. After chromatographic separation of pyrimidine tracts (isostichs or isoplyths) according to the number of contiguous pyrimidines, semilog plots of tract frequency vs. the number of contiguous pyrimidines were linear, plots for DNA from several sources differed from one another, and all deviated significantly from randomness. Similar semilog plots for coding sequences among 60 mammalian genomes or 28 rat tissue genomes were intermediate among slopes for isolated DNA. Individual isostichs were hydrolyzed, and their constituent pyrimidine bases were analyzed by high-pressure liquid chromatography. Among isostichs from isolated DNAs, the distribution of Thy and Cyt contents differed markedly from the distribution of 5-methylcytosine (5-Me-Cyt); e.g., although isostich 1 contained 45-49% of 5-Me-Cyt, amounts of Thy or Cyt did not exceed 25%. Semilog plots of normalized values for tract frequency or the content of 5-Me-Cyt vs. isostich number were essentially superimposable; thus, among the first five pyrimidine tracts of a particular tissue or E. coli DNA, the number of tracts per 5-Me-Cyt moiety was essentially constant. The data showed that 5-Me-Cyt and/or dCyd-dGuo dinucleotides have a distribution throughout DNA structure that superimposes the distribution of pyrimidine tract frequency and suggests that regulatory 5-Me-Cyt moieties are principally located at 3' termini of pyrimidine tracts.

Assessment of salvage pathways utilized for incorporation of exogenous pyrimidine nucleosides into DNA of guinea pig lymphocytes stimulated by Con A

“…To determine which DNA bases were labeled by a particular radioprecursor, isolated DNA was hydrolyzed to purine and pyrimidine bases in 98% formic acid and the bases separated by HPLC on a Supelcosil LC-18-DB column (Supelco, Bellefonte, PA) using isocratic elution with a buffer containing 5 mM KHzPO4 (pH 4.0) and 0.1% tetrahydrofuran [15]. A typical analysis consisted of injecting 2000-8000 cpm of DNA hydrolysate, collecting 0.5 ml fractions and determining radioactivity content by liquid scintillation counting.…”