Stimulated Normal Human Lymphocytes Contain a Ribonuclease-Sensitive DNA Polymerase Distinct from Viral RNA-Directed DNA Polymerase

Abstract

Ribonuclease-sensitive DNA synthesis is demonstrated in a cytoplasmic particulate fraction of normal human blood lymphocytes stimulated with phytohemagglutinin, but not in unstimulated lymphocytes.DNA polymerase purified from this fraction does not transcribe the heteropolymeric regions of 70S RNA from RNA tumor viruses, thus distinguishing this enzyme from the RNA-directed DNA polymerase (reverse transcriptase) found in oncogenic RNA viruses and human leukemic cells.After the discovery of RNA-directed DNA polymerase (reverse transcriptase) in RNA tumor viruses (1-3), we undertook a search in both normal and neoplastic cells for this or a similar enzyme (4-6). Temin and others have suggested that RNA-instructed DNA synthesis may play a role in normal cellular differentiation, gene amplification, and in the secondary immune response (3, 5-9). The possible location of RNAdirected DNA polymerase in the cytoplasm of cells as either a viral or subviral particle, associated with an RNA template/primer, prompted a search for RNase-sensitive DNA synthesis directed by endogenous template/primers in highspeed, partially-purified "pellet" fractions from cytoplasms of mammalian cells (10, 11). Coffin and Temin (10) described such an endogenous RNase-sensitive DNA polymerase reaction in the high-speed cytoplasmic "pellet" of both virusinfected and uninfected rat tissue-culture cells. Ackermann et al. (11) described a similar reaction in a continuous cell line established from leukocytes of a patient with leukemia, but this cell line was thought to contain type-C viruses. Recently, RNase-sensitive DNA synthesis has been found in cytoplasmic fractions of fresh cells, including normal chick-embryo cells (12) and human leukemic blood leukocytes (13,14).Recently, reports have appeared showing that RNA may be a primer for DNA-directed DNA synthesis in bacterial (15, 16) and mammalian (17, 18) systems. Therefore, sensitivity of DNA synthesis to RNase in a complex system such as these cytoplasmic "pellets" (10-14) could be due to RNAprimed in contrast to RNA-directed DNA synthesis. One way to distinguish between a template and a primer role for RNA is to purify the DNA polymerase(s) from these "pellets" Abbreviation: PHA, phytohemagglutinin.

Reverse Transcriptase Activity: Increase in Marrow Cultures from Leukaemic Patients in Relapse and Remission

“…Second, enzyme activity was found in marrow cells obtained from patients in remission and increased following culture, often reaching the levels seen in patients in relapse. The differential counts on these remission marrows were close to those of normals; accordingly, our findings are consistent with the view that presence of the enzyme activity cannot be attributed to the presence of large numbers of undifferentiated cells (Bobrow et al, 1972).…”

Characterization and distribution of nucleic acid sequences of a novel type C retrovirus isolated from neoplastic human T lymphocytes.

“…The T cells of origin for HTLV (designated HUT 102 and originating from the lymph node of the T-cell lymphoma patient), the methods of viral harvest and purification, and the methods for short-term (72 hr) culture of phytohemagglutinin-stimulated peripheral blood lymphocytes from pooled normal donors have been described (11)(12)(13) After termination of the reaction with NaDodSO4, cDNA was purified by alkaline hydrolysis (0.3 M KOH, 10 min, 100°C), organic solvent extraction, and precipitation with cetyltrimethylammonium bromide as described (16). In some cases, [3H]cDNA was prepared in reconstituted rather than endogenous reverse transcriptase reactions.…”

Ribonuclease‐sensitive DNA polymerase activity associated with particles distinct from A type and C type viral particles in murine myeloma tumor cells

“…However, it cannot be excluded that the formation of these particles is somehow related to the information contained in the viral genome, which is present in both fibroblastic and epithelioid cells. Another possiblity is that these particles are present in all rapidly dividing cells, as suggested by the results of the Temin's group, and by the fact that similar particles are also observed by Borrow et al in phytohemagglutinin-stimulated lymphocytes [19]. A possible role of such particles and of their apparent RNA-directed DNA polymerase was suggested by Temin [20,21 ] in the discussion on the protovirus hypothesis: they could be involved in some processes of normal cellular differentiation.…”