Characterization of Cytoplasmic DNA of Mouse‐Myeloma Cells

Abstract

Cytoplasmic DNA from mouse myeloma cells comprised between 1 % and 2 % of the total cellular DNA. Detergent-prepared cytoplasmic lysate consisted mainly of 8-S and 22-S species. While these DNA species were present in the 13 000 x g pellet of the detergent-prepared cytoplasmic lysate, only the light DNA species was present in the 13000 x g supernatant fraction. In neutral CsCl gradients the DNA of both cytoplasmic fractions had a buoyant density of 1700 g/cm3, which is identical to that of nuclear DNA.The similarity between the cytoplasmic and nuclear DNA was also demonstrated by analysis on alkaline CsCl gradients. A small proportion of closed-circular DNA, presumably of mitochondrial origin, was demonstrated only in cytoplasmic fraction obtained from mechanically disrupted cells and not in detergent-prepared cytoplasmic lysate. It was found that poly(A)-containing mRNA and 28-S ribosomal RNA hybridized to about the same extent to the cytoplasmic DNA as compared to nuclear DNA. The results indicate that most of the cytoplasmic DNA in myeloma cells is similar to nuclear DNA and does not consist of mitochondrial DNA. Several classes of cytDNA, different from mtDNA, have been demonstrated in mammalian cells. Rapidly labeled DNA has been found to be associated with the microsomal subcellular fraction [l-51 and to form DNA protein particles in various cell types [5,6]. Evidence has been presented indicating that this DNA was leaking from nuclei damaged in the course of cell fractionation [4,7]. This possibility, however, was not supported by the finding that microsomal DNA has a higher turnover rate compared to that of nDNA [l-4,8], and that the two DNAs can be differentially inhibited by inhibitors of DNA synthesis [9,10]. A second type of cytDNA has been reported to be associated with the outer cell membrane of a human lymphoid cell line [ll]. In contrast to microsomal DNA this DNA was labeled at a lower rate than nDNA [12,13]. More recently Smith and Vinograd [14] have demonstrated a small polydisperse closed-circular DNA in the cytoplasm of HeLa and other mammalian cells, distinguishable from mtDNA by size and buoyant density. The function of these cyt-DNA species is still obscure. It has been suggested that microsomal DNA has an informational role [5] and that membrane-associated DNA may participate in the transfer of the antigenic signal from the outer membrane of the Abbreviations Cytoplasmic DNA, cytDNA; nuclear DNA, nDNA; mitochondria1 DNA, mtDNA. lymphoid cell to its nucleus [ll]. If any of the cytDNA species described had an informational role, it should have contained only a small proportion of the total nucleotide sequences of the genome. Thus, microsomal DNA does not seem to be informational, since it was shown to resemble nDNA closely in its base composition [8] and reassociation rate [15]. On the other hand, even though the lymphoid cell membrane DNA had the same buoyant density as nDNA it was shown to have quite different reassociation kinetics indicating the presence of different frequencies of c...

Mammalian Argonaute-DNA binding?

“…Further studies showed that cytoplasmic DNA represents 1-2% of total cellular DNA in myeloma cells. Structurally, more than half of cytoplasmic DNA had a sedimentation constant of 6-8S, the remaining being more heterogeneous (12S to 40S) [ 26 ]. Cytoplasmic non-mitochondrial DNA with size in the range of 50-500 bp was observed in mouse L929 and Ehrlich ascites tumor cells, and some cytoplasmic DNA sequences, when cloned and transferred to non-tumor cells, could immortalize recipient cells [ 27 ].…”