A New Synthetic RNA-Dependent DNA Polymerase from Human Tissue Culture Cells

Abstract

Two DNA polymerases that can copy synthetic RNA polymers are present in human tissue culture cells. These enzymes which have each been purified about 500-fold, are present in both HeLa cells, which are derived from a cervical carcinoma, and in WI-38 cells, a normal diploid strain originating from human embryonic lung tissue. These synthetic RNA-dependent DNA polymerases are identified by their ability to copy efficiently the ribo strand of synthetic oligonucleotide-homopolymer complexes, and differ in this respect from the known DNA-dependent DNA polymerases found in HeLa cells. The template requirements of these new DNA polymerases resemble that of the RNA-dependent DNA polymerases of the RNA tumor-viruses.Since the discovery by Temin and Mizutani (1) and Baltimore (2) of an enzyme that could use endogenous viral RNA as a template for the synthesis of DNA, several studies have been published concerning the characteristics of this new type of DNA polymerase.Initially this enzyme was found in a wide variety of oncogenic viruses containing RNA (3). For this reason it was thought that this type of DNA polymerizing activity was unique to the RNA tumor-viruses. However, different investigators claim to have shown the presence of RNA-dependent DNA polymerase (JR-DNA polymerase) activity in several types of cells (4-8). None of these reports has clearly established the presence of this enzyme in normal eukaryotic cells, since the criteria used to determine the enzymatic activity are not readily accepted as unique for RNA-dependent DNA polymerases. As has been shown by Goodman and Spiegelman (9), the main difference between the RNA-dependent DNA polymerase from avian myeloblastosis virus and the DNA-dependent DNA polymerase (D-DNA polymerase) triphosphates, dithiothreitol, and salmon-sperm DNA were obtained from Calbiochem. Whatman microgranular DEAEcellulose, DE52, 1.0 meq/g of dry weight, and phosphocellulose, P-11, 7.4 meq/g of dry weight were obtained from Reeve-Angel. Poly(rA), poly(dT), poly(rU), poly(dT) poly-(rA), and poly(rA) * poly(rU) were obtained from Miles Laboratories, Elkhart, Ind. The oligodeoxynucleotides (dT),2-8, and (dA)2-is were purchased from Collaborative Research, Waltham, Mass. The oligonucleotide (rU)29 was provided by Dr. S. Pestka (Roche Institute of Molecular Biology); it was prepared by treatment of poly(rU) with pork-liver nuclease (obtained from Dr. Leon Heppel) and subsequent chromatography on a Sephadex column. The average chain length of this oligonucleotide preparation, as determined by the ratio of terminal phosphate to total phosphate content, was 29. Oligodeoxynucleotides were annealed (10) to homopolymers at a proportion of three parts of oligodeoxynucleotide to seven parts by weight of homopolymer. Activated salmon-sperm DNA and activated HeLa DNA were prepared by treatment of the DNA with pancreatic DNase (11).Growth of Cells. HeLa S-3 cells were grown in suspension cultures at 37°C in F-13 medium (Gibco) supplemented with 5%/0 fetal-calf serum (Gibco), and were harvest...

Terminal Deoxynucleotidyl Transferase in a Case of Childhood Acute Lymphoblastic Leukemia

“…The third enzyme, assayed by measuring poly(dT) synthesis stimulated by poly(A) . oligo(dT) in the presence of Mn++ (6,13), is called DNA polymerase A. It is present in most cells that we have examined, and has been reported by many investigators (13)(14)(15).…”

“…This enzyme eluted on the trailing edge of the DNA polymerase C peak, as it does in all other human cell preparations we have investigated. It is separable from DNA polymerase C by use of other fractionation methods (6).…”

DNA polymerase activity of cultured rheumatoid synovial cells

“…Poly (rA):oligo (dT)lZ-lS contains an RNA template which is copied by both oncornavirus and normal cellular DNA polymerases, and hence is a nonspecific measure of RNA-directed DNA polymerase activity (RDDP). Poly (rC):oligo (dG)12-18 contains an RNA template which has been shown to be highly specific for the oncornavirus RDDP (6,8,12).…”

“…Enzyme activity w i t h these synthetic templates is several fold greater t h a n w i t h n a t u r a l nucleic acid heteropolymers, allowing assay of small q u a n t i t i e s of enzyme. In addition, t h e preferential utilization of p a r t i c u l a r synthetic templates by individual polymerases provides a means of characterization of these enzymes (6,8,12).…”

Histamine and Radiation-Induced Taste Aversion Conditioning

“…suggested for DNA polymith high effi-ONA well (8). R-DNA poropensity for cleotides, but as to its abililymerase-y is ,ht >100,000, taming compolymerase enzyme sepa-Histamine and Radiation-Induced Taste Aversion Conditioning ); it is named Levy, Carroll, Smith, and Hofer (1) ren, which is so ported that rats treated with an antihistamine do not subsequently develop radid y have been ation-induced taste aversions, and sugblications by gested that radiation-induced histamine production is the critical event responsible 4A polymer-for the formation of radiation-induced ng and will be taste aversions.…”