Labeling and selective recovery of newly synthesized viral DNA from simian virus 40-infected cells incubated with inorganic thiophosphate.

Abstract

African green monkey CV-1 cells infected with simian virus 40 (SV40) were incubated in the presence of sodium [MS]thiophosphate. The viral DNA was prepared and the newly synthesized SV40 DNA molecules containing phosphorothioate groups were selectively recovered by affinity chromatography on organomercurial-Sepharose columns. The thio-derivatized viral DNA occurred in both superhelical (form I) and relaxed circular (form II) configurations, and it gave the expected cleavage products upon digestion with the restriction endonucleases Kpn I and HindIl. Enzymatic digestion of 35S-labeled SV40 DNA to its component nucleotides and chromatographic separation of the resultant 5'-labeled phosphorothioate esters established the presence of the label in all four major nucleotides, but mainly in thymidylic acid. The ability to label and recover selectively the newly synthesized polynucleotides by incubating cells with inorganic thiophosphate complements earlier findings [ The method has been employed to study the initiation of 5S RNA in myeloma nuclei (S), ofpreribosomal RNAs in Physarum polycephalum (6) and Xenopus laevis oocyte nuclei (7), and of pre-tRNAs in yeast nuclei (8).Thio-derivatized deoxynucleoside triphosphates substituted in the a position have been employed as precursors for the in vitro synthesis ofviral DNAs (9, 10), and they permit an analysis of the stereochemistry of the DNA polymerase reaction (11). Some interesting recent applications include the synthesis of a 'S-labeled cDNA of rabbit globin mRNA (12) and the use of dATP [a-35S] for the nick-translation of plasmid DNA (12).While it is clear that the phosphorothioate analogues of nucleoside and deoxynucleoside triphosphates have many important applications, such compounds are not effective substrates for the in vivo synthesis of polynucleotides, due to restricted permeation through the cell membrane. What is needed is a thio-derivatized precursor that can freely enter the nucleotide pools of living cells. We have recently shown that [3S]thiophosphate fulfils this requirement and effectively labels ATP, GTP, and other nucleotides of HeLa cells (13). The subsequent transfer of thiophosphate to nuclear proteins permits their purification by Hg-affinity chromatography and facilitates the analysis of different sites of modification within the polypeptide chain.Given the evidence that inorganic thiophosphate readily enters the ribonucleotide pools of cultured cells, we decided to test whether deoxyribonucleotide pools were also labeled to a degree permitting the isolation of thio-derivatized DNA. The experiments to be described indicate that [3S]thiophosphate can be used to label the DNA of simian virus 40 (SV40) in infected CV-1 cells and that the substitution permits purification of new SV40 molecules by Hg-affinity chromatography. MATERIALS AND METHODSThe African green monkey cell line CV-1 and SV40 were the generous gifts of M. T. Hsu of the Rockefeller University. The growth and maintenance of the cell line and propagation of SV40 were carrie...

Interaction of tRNAs and of phosphorothioate-substituted nucleic acids with an organomercurial. Probing the chemical environment of thiolated residues by affinity electrophoresis

“…The strong interaction of sulfur-containing nucleic acids with organomercurials has been the subject of numerous reports [e.g., Scheit and Faerber (1973), Sunshine and Lippard (1974), and Maguire (1976)], while the use of mercurated T This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 206) to Prof. H. Kossel. column materials for the isolation of sulfur-containing macromolecules is well documented (Melvin et al, 1978;Sun & Allfrey, 1982;Zhang et al, 1984) and the matrices for such batch procedures are commercially available. The wide range of chemical environments available to the sulfur atom provides a range of compounds whose different reactivity toward mercury might be expected to permit high-resolution fractionation by affinity electrophoresis.…”