Characterization of rapidly labeled detergent-soluble DNA in murine splenocytes.
Freshly prepared spleen cells from concanavalin A stimulated mice incorporate [3H]thymidine into DNA which can be recovered in detergent-soluble (NP40) and detergent-insoluble forms. The presence of detergent-soluble forms occurs despite the fact that the cells are lysed at 4 degrees C in the presence or absence of 25 mM ethylenediaminetetraacetic acid. After a 2-h pulse with [3H]thymidine, the detergent-soluble fraction contains about 1-3% of the total cellular DNA but 25% of the total labeled high molecular weight material. Since the specific activity of the extensively purified DNA from the detergent-soluble fraction is considerably higher than that of chromosomal DNA, it meets the criteria for being metabolically active. We propose the name "MADS" DNA for metabolically active detergent-soluble DNA. MADS DNA has a density of 1.699 g/mL on cesium chloride gradients and a slightly higher G + C content than chromosomal DNA as determined by high-pressure liquid chromatography. Electrophoresis using native or denaturing agarose gels resolves MADS DNA into discreet sizes between 200 and 4500 base pairs. Nuclease S-1 treatment of native MADS DNA does not alter the size distribution as resolved by means of gel electrophoresis under denaturing conditions. Therefore, MADS DNA is not a collection of single-stranded Okazaki fragments. Southern blot analysis reveals that mitochondrial DNA is a minor component of higher molecular weights above the bulk of the DNA visualized either by staining with ethidium bromide or by incorporation of [3H]thymidine. Inhibitors of ribonucleotide reductase or DNA polymerase alpha inhibit incorporation of [3H]thymidine into MADS DNA, and hence chromosomal DNA synthesis is required for MADS DNA production. Since Southern blot analysis also reveals homology of larger fragments with the 32P-labeled 200 base pair fragment, the presence of repetitive sequences is suggested.
D000818:Animals; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004261:DNA Replication; D003902:Detergents; D007700:Kinetics; D008214:Lymphocytes / Q000378:metabolism*; D008297:Male; D051379:Mice; D009693:Nucleic Acid Hybridization; D017830:Octoxynol; D011092:Polyethylene Glycols; D014176:Protein Biosynthesis; D012995:Solubility; D013154:Spleen / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D014316:Tritium
D003902:Detergents; D014316:Tritium; D011092:Polyethylene Glycols; D017830:Octoxynol; D004247:DNA; C010615:Nonidet P-40; D013936:Thymidine