Strandedness and complementarity of DNA from long-term RNA-dependent DNA polymerase reactions of Soehner-Dmochowski murine sarcoma virus.
The DNA product of the endogenously instructed RNA-dependent DNA polymerase reaction of murine sarcoma virus continued to be synthesized for as long as 64 h in the presence of 0.008% Triton X-100. Higher detergent concentrations and actinomycin D inhibited DNA product synthesis. The DNA product from long-term polymerase reactions consisted of small DNA fragments as shown by sedimentation in alkaline sucrose gradients. The enzymatic DNA product was separated into a slow sedimenting fraction and a fast sedimenting fraction by rate-zonal centrifugation. Fast sedimenting DNA was the predominant fraction made in viral polymerase reactions containing 262 mM NaCl. By using a combination of S-1 nuclease and pancreatic RNase A, the amount of single-stranded DNA, double-stranded DNA, and DNA-RNA hybrid present in the slow-sedimenting and fast-sedimenting fractions was determined. Under standard polymerase conditions of 70 mM NaCl, single-stranded DNA was the major form of DNA found in both fractions. In contrast, the prevalent form of DNA made in the presence of 262 mM NaCl was DNA-RNA hybrid. Hybridization studies in which either S-1 nuclease or pancreatic RNase A was used to measure hybrid formation demonstrated not only that the DNA product was complementary in base sequence to the RNA genome, but also that at least 79 to 84% of the RNA genome was transcribed into complementary DNA.
D000818:Animals; D001230:Aspergillus; D001483:Base Sequence; D002460:Cell Line; D002501:Centrifugation, Zonal; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000302:isolation & purification; D004279:DNA, Viral / Q000032:analysis* / Q000037:antagonists & inhibitors; D003609:Dactinomycin / Q000494:pharmacology; D003851:Deoxyribonucleases; D017988:Gammaretrovirus / Q000201:enzymology* / Q000302:isolation & purification; D006868:Hydrolysis; D051379:Mice; D009693:Nucleic Acid Hybridization; D010179:Pancreas / Q000201:enzymology; D010727:Phosphoric Diester Hydrolases; D012367:RNA, Viral; D051381:Rats; D012260:Ribonucleases; D012965:Sodium Chloride / Q000494:pharmacology; D013501:Surface-Active Agents; D013696:Temperature; D013997:Time Factors; D014158:Transcription, Genetic; D014316:Tritium
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D012367:RNA, Viral; D013501:Surface-Active Agents; D014316:Tritium; D003609:Dactinomycin; D012965:Sodium Chloride; D004254:DNA Nucleotidyltransferases; D003851:Deoxyribonucleases; D012260:Ribonucleases; D010727:Phosphoric Diester Hydrolases