New procedure for isolation of Rous sarcoma virus-specific RNA from infected cells.
The use of mercurated "strong stop" complementary DNA (complementary to the 5'-terminal 101 nucleotides of Rous sarcoma virus RNA) in the isolation of virus-specific RNA from infected chicken embryo fibroblasts is described. Strong stop Rous sarcoma virus complementary DNA was mercurated chemically, and, as a result of the low complexity of this DNA, short hybridization times (up to 15 min) and heating in the absence of formamide were found to be adequate conditions for the isolation of virus-specific RNA. The purity of the isolated RNA was demonstrated by analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. The isolated RNA could be translated in the in vitro protein synthesis system derived from rabbit reticulocytes, and an analysis of polypeptides programmed by isolated RNA before and after immunoprecipitation further demonstrated both the purity of the isolated mRNA and the quantitative nature of the isolation procedure.
D000818:Animals; D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D001358:Avian Sarcoma Viruses / Q000032:analysis* / Q000378:metabolism; D002460:Cell Line; D002642:Chick Embryo; D004279:DNA, Viral / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005347:Fibroblasts; D008628:Mercury / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification*; D014764:Viral Proteins / Q000096:biosynthesis
D004279:DNA, Viral; D012367:RNA, Viral; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; D008628:Mercury