In vitro replication of tobacco mosaic virus RNA in tobacco callus cultures: solubilization of membrane-bound replicase and partial purification.

摘要: A fraction containing membrane-bound tobacco mosaic virus RNA replicase was isolated form virus-infected callus cultures. The activity reached a maximum 60 h after inoculation and then declined. enzyme insensitive to actinomycin D DNase. corresponding from healthy contained essentially no activity. viral synthesis in vitro proceeded linearly for 30 min required the four nucleotide triphosphates Mg2+ ions. Mn2+ poor substitute Mg2+. During product at least 70% resistant RNase 2X SSC (0.15 M NaCl plus 0.015 sodium citrate), but completely digested by 0.1X SSC. Analysis of polns) that appeared be replicative partially RNase-resistant structure similar intermediate form. Washing with Mg2+-deficient buffer solubilized enzyme. solubulized further purified DEAE-Sephadex column chromatography. DEAE-purified nearly dependent upon on sucrose gradient revealed double-stranded sedimentation 16S smaller heterogeneous RNase-sensitive products.