The Friend spleen focus-forming virus (F-SFFV) released from cultured erythroleukemia cells (cell line F4-6/K) was cloned free of its helper lymphatic leukemia virus (F-MuLV). After allowing adsorption to Sc-1 fibroblasts at a low multiplicity of infection, the cells were seeded individually into wells of a microtiter test plate and the resulting colonies were grown into large cultures. Among 14 of these cell cultures that have been analyzed thoroughly, 6 contained F-SFFV alone, 1 contained F-MuLV plus F-SFFV, and 7 were uninfected. Each of the Sc-1 cell lines which had been infected with cloned F-SFFV contained a glycoprotein with an apparent molecular weight of 55,000 (gp55) that was absent from the cell lines that lacked F-SFFV. gp55 was also present in Friend erythroleukemia cells and in fibroblasts infected with an F-SFFV that had been doubly cloned in another laboratory. These results indicate that gp55 is encoded by the F-SFFV genome. gp55 has the following additional properties. It can be immunoprecipitated with antiserum made to the F-MuLV virion envelope glycoprotein (gp75). Its unglycosylated polypeptide, formed in cells treated with 2-deoxy-D-glucose, has a molecular weight of approximately 45,000. Its tryptic peptide map contains peptides in common with F-MuLV gp75 but it also contains unique peptides. It appears to be absent or present in only low concentrations in erythroleukemia cell plasma membranes as determined by lactoperoxidase-catalyzed iodination, and it accumulates intracellularly in large amounts. In addition, it is absent from released virions. The majority of the cellular gp55 has an isoelectric point of 8.5 to 9.0. These results are consistent with the idea that an env gene recombination event was involved in the origin of F-SFFV.

Synthesis and post-translational processing of murine leukemia virus proteins were analyzed in a murine cell line (Eveline) that produces large amounts of Friend lymphatic leukemia virus. Immunoprecipitation of L-[35S]methionine-la

We have extended our permeable cell system for measuring DNA excision repair [Roberts, J. D., & Lieberman, M. W. (1979) Biochemistry 18, 4499-4505] so that steps of the repair process, beginning with incision and extending at least through the "rearrangement" of repaired nucleosomes which follows repair synthesis, all take place in permeable cells. In the revised protocol, human fibroblasts are made permeable, damaged with UV or chemicals in suspension, and incubated with a reaction mix containing ATP and the four deoxyribonucleoside triphosphates, one of which is labeled with 32P. By reducing the exogenous dNTP concentration to 3 microM and including 15 mM KCl in the reaction mixture, we have greatly reduced background incorporation in undamaged cells without significantly reducing repair synthesis. This permits us to measure repair synthesis without separating it from replicative synthesis by isopycnic centrifugation. Repair synthesis in this system is very similar to that occurring in intact cells: in response to DNA damage, nucleotides are incorporated into DNA of parental density (when analyzed by the BrdUrd density shift technique), incorporation increases with increasing DNA damage, synthesis is dependent on the presence of all four dNTPs, and the system accurately reflects the genetic UV repair deficiency of xeroderma pigmentosum (XP) cells. Furthermore, as has been observed in intact cells, repair-incorporated nucleotides in these permeable cells are initially overrepresented in staphylococcal nuclease sensitive regions of chromatin and are subsequently redistributed to give a nearly uniform distribution between nuclease-sensitive and -resistant regions. The UV dose curve of permeable cells differs somewhat from that of intact cells; however, the dose differs somewhat from that of intact cells; however, the dose curve for permeable cells treated with N-methyl-N-nitrosourea is very similar to that of intact cells. Repair synthesis in UV-damaged, permeable normal and XP cells is stimulated by addition of Micrococcus luteus UV endonuclease, indicating that the damaged DNA is accessible to exogenous repair enzymes and suggesting that incision, or an obligatory preincision step, is rate limiting for excision repair in these permeable cells. Repair synthesis in this system is inhibited by aphidicolin, but not by high levels of dideoxy-TTP, suggesting involvement of DNA polymerase alpha in excision repair. Novobiocin is also inhibitory alpha and the HeLa cell type II DNA topoisomerase.

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It is well established that DNA replication and ultraviolet-induced DNA repair synthesis in mammalian cells are aphidicolin-sensitive and thus are mediated by one or both of the aphidicolin-sensitive DNA polymerases, alpha and/or delta. Recently, it has been shown that DNA polymerase delta is much more sensitive to inhibition by the nucleotide analogue 2',3'-dideoxythymidine 5'-triphosphate (ddTTP) than DNA polymerase alpha but is less sensitive than DNA polymerase beta [Wahl, A. F., Crute, J. J., Sabatino, R. D., Bodner, J. B., Marraccino, R. L., Harwell, L. W., Lord, E. M., & Bambara, R. A. (1986) Biochemistry 25, 7821-7827]. We find that DNA replication and ultraviolet-induced DNA repair synthesis in permeable human fibroblasts are also more sensitive to inhibition by ddTTP than polymerase alpha and less sensitive than polymerase beta. The Ki for ddTTP of replication is about 40 microM and that of repair synthesis is about 25 microM. These are both much less than the Ki of polymerase alpha (which is greater than 200 microM) but greater than the Ki of polymerase beta (which is less than 2 microM). These data suggest that DNA polymerase delta participates in DNA replication and ultraviolet-induced DNA repair synthesis in human cells.