A method is given for small-scale preparation of DNA from 1.0-1.5g of adult rat tissues. The product from brain or liver is characterized by base ratios and phosphorus content which accord with reported values for rat tissue. It is reasonably free of RNA, protein and glycogen. It contains 5-hydroxymethylcytosine at a content of about 15% of the total cytosine bases present. 5-Hydroxymethylcytosine is also demonstrable in mouse and frog brain DNA and in the crude cytidylic acid fractions obtained from RNA hydrolysates of rat brain and liver. 5-Hydroxymethylcytosine is identified by paper chromatography, u.v. spectra in acid and alkaline solutions and by its conversion into 5-hydroxymethyluracil.
The mol percentage of 5-hydroxymethylcytosine is 2.2 times greater in the adult than in 2-day-old rat brain DNA. The concentration of 5-hydroxymethylcytosine falls in corresponding liver DNA preparations. This normal increase in brain 5-hydroxymethylcytosine is abolished in rats placed on an 8 %-protein diet 5 days after birth.The pyrimidine 5-hydroxymethylcytosine has been reported to characterize a central-nervous-system DNA species that is enzymically and chemically labile (Penn et al., 1972). This DNA was implicated in higher functions of the central nervous system, presumably through modulation of synaptic activity by a fraction localized in the synaptosomal mitochondria (N. W. Penn, S. Schell, R. Suwalski & K. Bojanowski, unpublished work). In support of this interpretation, further study has demonstrated that the effects of barbiturate, alcohol and morphine are antagonized by DNA pyrimidines and their metabolically allied cofactors (Penn, 1974(Penn, , 1975a. Time-intervals involved in reversal of drug action, barbiturate binding to DNA fractions and the specificity for DNA components as antagonists suggest a direct interaction between these drugs and a DNA receptor(s).The acute actions of these drugs are mediated primarily or in part at the central-nervous-system synapse. It thus appeared that the DNA species characterized by 5-hydroxymethylcytosine, or the concentration of this base in DNA, might increase as the synaptic architecture of the brain was completed. We therefore investigated hydroxymethylcytosine concentrations in newborn and adult, normal and retarded rat brain DNA, by using liver DNA as a reference preparation. Materials and MethodsThe 8 % (low-protein) diet for rats was purchased from ICN Life Sciences, Cleveland, OH, U.S.A. The original procedure for DNA isolation was used (Penn et al., 1972) 1972), and homogenized in solution A (Solution A/ brain = 10:1, v/w). Brains from 2-day-old rats were similarly processed, combining tissue from two newborn animals for each sample. Livers from four 2-day-old animals, similarly prepared, were combined for each determination. To retard development, 5-day-old rats were placed on an 8%-protein diet, separated from the mother 2 weeks later and maintained on the diet until they were killed 2 months after birth. Animals from three litters were used.The individual nucleotide bases were determined after formic acid hydrolysis. Appropriate portions were chromatographed in duplicate in propan-2-ol-/12M-HCl/water (170:41:39, by vol) (Wyatt, 1951) for precise evaluation of base ratios. Cytosine and 5-hydroxymethylcytosine were determined as total cytosines by this system, which does not resolve these pyrimidines. The ratio between the two componentswasdeterminedbyduplicatetwo-dimensional chromatographic runs in sodium acetate (0
1. A procedure is given for spectrophotometric analysis of rat brain DNA after its resolution into component bases. Amounts of tissue in the range 50-100mg. can be used. 2. The amount of DNA obtained by the present method is 80% greater than that reported for rat brain by a previous procedure specific for DNA thymine. Identity of the material is established by the base ratios of purines and pyrimidines. The features responsible for the higher yield are the presence of dioxan during alkaline hydrolysis of tissue, the determination of the optimum concentration of potassium hydroxide in this step and omission of organic washes of the initial acid-precipitated residues. 3. The requirement for dioxan during alkaline hydrolysis suggests a possible association of brain DNA with lipid. The concentration of potassium hydroxide that gives maximum yield is 0.1m, indicating that there may be internucleotide linkages in this DNA that are more sensitive to alkali than those of liver or thymus DNA. 4. This procedure gives low yields of DNA from liver. It is not suitable for analysis of the DNA from this tissue.
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