Different Utilization of Deoxycytidine As DNA-Thymine in Lymphoid Tissues of the Mouse

CELL STRUCTURE AND FUNCTION 3, 255~257 (1978) C by Japan Society for Cell Biology

Different Utilization of Deoxycytidine as DNA-Thymine in Lymphoid Tissues of the Mouse

Kiyohiro Hamatani and Minoru Amano* Department of Zoology, Faculty of Science, Faculty of Integrated Arts and Sciences*, Hiroshima University, Hiroshima 730, Japan

ABSTRACT. Small pieces of tissues were cut and homogenized in an appropriate volume of 2 M NaCl. Pure, highly polymerized DNA was extracted after RNase and protease treatment from the homogenate of lymphoid tissues of mouse. The extracted DNA contained no detectable RNA, and the yields ranged from 30 % to 70 % of DNA in the starting material. Using pure DNA extracted by this method, the rate of [G-3H]deoxycytidine incorporation into cytosine and thymine of DNA in thymus was different from that in mesenteric lymph nodes in mouse after administration of [G-3H]deoxycytidine. Thus, the utilization of deoxycytidine as DNA-thymine was differed between the thymus and mesenteric lymph nodes in the mouse.

There are many methods of DNA extraction from the different tissues of higher organisms (5, 6, 7, 8, 12), but most procedures have been designed to extraction of DNA from large pieces of tissues (5, 6, 7). At present, there are few methods for the preparation of highly polymerized DNA from very small tissues such as a piece of the mesenteric lymph nodes (M.L.N) of mice. It was necessary to establish a method for pure DNA extraction from small pieces of tissue and to examine the rate of conversion of deoxycytidine to thymidine monophosphate used as DNA-thymine in different lymphoid tissues of the mouse (1, 2, 10, 11). To facilitate deproteinization and to increase the efficiency of DNA extraction, small pieces of tissue were homogenized in 2 M NaCl, and the pure DNA was extracted by a modification of Marmur's method (9), i.e. in combination with enzyme treatment. Moreover, it was important to determine the most appropriate concentration of homogenate. By considering these points, an adequate amount of highly polymerized DNA could be extracted from the M.L.N of the mouse. M.L.N, with about 25.7 mg in wet weight, were finely cut with a pair of scissors, then homogenized in 1.3 ml of 2 M NaCl containing 0.3 % sodium dodecyl sulfate (Sigma Chemical Company St. Louis, USA) in a Teflon-glass homogenizer. The tissue was sufficiently homogenized to break all the cells. An equal volume of chloroform- isoamyl alcohol (24 : 1, v/v) was then added to the homogenate and the mixture was shaken vigorously, after which it was centrifuged at 4,500 xg for 10 min. The inter- phase and chloroform-isoamyl alcohol phase were mixed with 1.3 ml of 2 M NaCl and homogenized again. After a similar centrifugation, the combined aqueous phase was mixed with 2 volumes of cold ethanol. Fibrous DNA was spooled on a glass rod, then dissolved in 1.0 ml of 5 mM EDTA and 50 mM acetate buffer solution (pH 4.5). At this step, it was necessary to dissolve the DNA in a smaller volume than that used

255 256 K. Hamatani and M. Amano for the first homogenate. This DNA solution was mixed with 0.1 ml of RNase T2

(20 units/ml : Sankyo Company Limited, Japan) and incubated for 3 hr at 37•Ž. After that, protease (Streptomyces Type VI : Sigma Chemical Company, St. Louis, USA) and 1.0 M Tris-HCl buffer (pH 7.6) were added to give final concentrations of 100

µg/ml and 0.1 M, respectively and the whole was incubated for 1 hr at 37•‹. After enzyme treatment, deproteinization was carried out twice with chloroform-isoamyl alcohol. The aqueous phase was made a final concentration of 0.14 M Na Cl and was mixed with 2 volumes of cold ethanol. The highly polymerized fibrous DNA was gathered by winding it on a glass rod and gently pressing out excess fluid. The extracted DNA was dissolved in 3.0 ml of distilled water. DNA and RNA determinations were carried out by the methods of Burton (4) and Webb (13), respectively. To determine the recovery of DNA, the total amount of DNA in other pieces of tissue was also measured. Yields ranged from 30 % to 70 % of the DNA in the starting material. In no instance did the DNA contain any detectable RNA. To analyze the base composition of the DNA, a sample of its solution was hydrolyzed in 6 N HCl at 100•Ž for 3 hr. After drying the material in a vaccum desiccator over P2O5, the residue was dissolved in very small quantity of 0.01 N HCl and spotted on a thin- layer chromatogram (Eastman chromatogram sheet, 13254 cellulose). Two dimensional thin-layer chromatography was used to separate the four bases. The first solvent used was 1-butanol : 15 N NH4OH : H2O (172: 8: 20, v/v) and the second was 2-propanol : concentrated HCl: H2O (70: 15: 15, v/v). The UV absorbing four spots were scraped off and each base was eluted with 0.01 N HCl. To analyze the rate of the conversion of deoxycytidine to thymidine monophosphate in different lymphoid tissue of the mouse, by the method described above, DNA was extracted from the thymus and from M.L.N after 6 hr or 24 hr of labeling with [G-3H]- deoxycytidine at 100 ƒÊCi per mouse (specific activity 20 Ci per m mol : New England Nuclear Corp. Boston, Mass). The radioactivity of the four bases in the extracted DNA was measured with Bray's scintillator solution (3) in a Beckman LS-100C scintillation counter. The ratio of 3H-thymine to 3H-cytosine was calculated from actual counts which ranged from 120 to 10,000 cpm when corrected for back-ground counts of 20-25 cpm. As shown in table 1, the ratio of 3H-thymine to 3H-cytosine in M.L.N was lower after 6 hr and 24 hr of labeling, respectively, than that in thymus. This indicates that the rate of [G-3H]deoxycytidine incorporation into the thymine

TABLE 1. RATIO OF 3H-THYMINE/3H-CYTOSINE IN DNA AFTER A [G-3H]DEOXYCYTIDINEINJECTION dCyd into DNA-T in Mouse Lymphoid Tissues 257

of the DNA in M.L.N is lower than that in the thymus. Both after 6 hr and 24 hr of labeling, the ratio of 3H-thymine to 3H-cytosine in the thymus of the mouse was higher than that in the thymus of the rat. The different ratios found in the mouse and in the rat (1) may reflect a difference in the activity of thymidylate synthetase and/or in the size of the thymidine pool. The different intensity of the incorporation of 3H- deoxycytidine or 3H-thymidine into lymphocytes in the thymus and M.L.N (1, 2, 10, 11) of the mouse and of the rat may be related to the rate of conversion of deoxycytidine to thymidine monophosphate. Thus, the difference in the ability to utilize deoxycytidine as DNA-thymine would affect the difference in the intensity in lymphocytes labeled with 3H-deoxycytidine in the thymus and in M.L.N. Therefore, thymic lymphocytes in the mouse and in the rat would be intensely labeled with 3H-deoxycytidine but not with 3H-thymidine.

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(Received for publication, June 30, 1978)