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A DNA-synthesizing system in vitro, using nuclei prepared by treatment of human lymphocytes with the detergent Brij 58, was developed. Nuclei from cultured lymphocytes synthesized DNA for as long as 5 hr, and required ATP, deoxynucleoside triphosphates, magnesium, and a calcium chelator. In nuclei from a partially synchronized line of cultured lymphocytes carrying several hundred copies of the Epstein-Barr viral genome, synthesis in vitro was predominantly viral in early S phase and cellular in late S phase. These and other data suggested that the DNA synthesis observed in vitro was predominantly replicative.)NA synthesis has been studied in a variety of eukaryotic cell types in culture, either in intact cells or in isolated nuclei, with both normal cell lines and cell lines infected by several DNA viruses, notably tumor viruses (for example, refs. 1-11). The study of both tumor virus and host DNA synthesis in virally transformed cells (as contrasted to productively infected cells) is made difficult by the problem of distinguishing the two processes. One cell system that would be suitable for such studies is human lymphocytes transformed by Epstein-Barr virus (EBV). There are available a wide range of lymphocyte lines that carry varying numbers of EBV genomes (12, 13). Furthermore, some of these are producer lines, i.e., a small portion of the population of cells is undergoing viral macromolecular synthesis at all times; others are nonproducer lines, in which viral DNA and antigen synthesis can be induced only by chemicals or superinfection (14-16).DNA synthesis in transformed lymphocytes is of particular interest for an additional reason. Normal peripheral lymphocytes are resting cells in terms of DNA synthesis, and EBV has been shown to cause them to undergo transformation into cells which now synthesize DNA and divide for an indefinite number of generations (17)(18)(19). It is, therefore, possible that the presence of the viral genome or some viral gene product (such as the Epstein-Barr nuclear antigen, EBNA) affects the control of host DNA synthesis in order for the cel] to assume the transformed state.Study of DNA synthesis in isolated nuclei has many advantages over its study in intact cells. However, lymphocyte nuclei are difficult to prepare in an active state after mechanical disruption in the Dounce homogenizer, the usual method for preparing nuclei, probably because their relatively large size renders them sensitive to damage. In the present paper a modified procedure for making lymphocyte nuclei in the presence of detergent without mechanical disruption of the cells (20, 21) is reported. These nuclei are active in a DNAsynthesizing system in vitro over a long period of time. Some characteristics of host and viral DNA synthesis in these lreparations will be reported.

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Low concentrations of adenine arabinoside inhibited growth of two Epstein-Barr virus producer cell lines in culture, while not significantly affecting a nonproducer cell line and a B-cell-negative line. These observations were extended to include freshly infected cells. Mitogen-stimulated human umbilical cord blood lymphocytes were unaffected by the drug at concentration levels that inhibited [3H]thymidine incorporation into the DNA of Epstein-Barr virus-stimulated cells. DNA synthesis in Epstein-Barr virus-superinfected Raji cells was also adversely affected by adenine arabinoside. However, these same low concentrations of adenine arabinoside in the triphosphate form produced less effect on DNA synthesis in nuclear systems and DNA polymerase assays than on growth or DNA synthesis in whole cells. Therefore the effects reported here of low concentrations of the drug on whole cells may be only in part related to DNA polymerase inhibition. The work reported here suggests that adenine arabinoside has multiple sites of action in infected cells. The synthesis of 9-fp-D-arabinofuranosyladenine (adenine arabinoside, or ara-A) as a potential anticancer agent was first reported in 1960 (18). Since then ara-A has been reported to have antitumor and antiviral effects for tissue culture cells and laboratory animals and in clinical use in treatment of certain virally caused diseases. Reports between 1964 and 1968 have shown that ara-A has specific antiviral effects in tissue culture for herpes, pox, and Rous sarcoma viruses (11, 29, 32; R. W. Sidwell, G. Arnett, and G. J. Dixon, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 7th, Chicago, Ill., Abstr. no. 64, 1967). Furthermore, inhibition of mammalian ribonucleotide reductase and DNA polymerase by ara-A were demonstrated (12, 22). Certain studies have shown that mammalian a polymerases are sensitive to ara-A (23-25;