Ribonucleotides in unintegrated linear spleen necrosis virus DNA

Abstract

The structure of unintegrated spleen necrosis virus DNA was characterized by using various chemical and enzymatic treatments in conjunction with denaturing gels and nucleic acid hybridization probes. Throughout the course of the viral infection, the predominant species of viral DNA was that of a linear double-stranded molecule containing ribonucleotides covalently joined to the DNA. The majority of both - and + strands were continuous. The ribonucleotide linkages appeared to be relatively short, and the base composition and distribution of the ribonucleotide linkages were heterogeneous. On the average, the - strand had fewer of the ribonucleotide linkages than did the strand. Viral DNA containing ribonucleotide linkages was infectious in DNA transfection assays. The structure of spleen necrosis virus DNA was different from that of Schmidt-Ruppin Rous sarcoma virus-D, and mixed infections demonstrated that the observed differences are a result of cis-acting functions.

Characterization of reticuloendotheliosis virus strain T DNA and isolation of a novel variant of reticuloendotheliosis virus strain T by molecular cloning

“…Analysis of unintegrated linear REV-T and REV-A DNAs. Chicken embryo fibroblasts grown on 100-mm culture dishes were infected with REV-T(REV-A) (REV-T,A) from spleen cell line [1][2][3][4][5][6] and REV-A at multiplicities of infection of approximately 1 and 0.1 PFU/cell, respectively. At 3 days after infection, the cells were lysed by the Hirt fractionation procedure (19).…”

“…DNA extraction and purification. Unintegrated viral DNA was prepared by the Hirt fractionation procedure, as previously described (5,19). Cellular DNA was prepared by lysing cells in 0.6% sodium dodecyl sulfate-10 mM EDTA and digesting the lysate with 100 ,ug of proteinase K per ml at 37°C for 24 h. The DNA was extracted with phenol and chloroformisoamyl alcohol as previously described (1).…”

“…Presoaking and hybridization of DNAs immobilized on nitrocellulose filters were performed as previously described (5). Filters were washed and exposed as previously described (5).…”

Establishment of infection by spleen necrosis virus: inhibition in stationary cells and the role of secondary infection

“…DNA extraction and purification. Unintegrated and integrated viral DNAs were prepared by the Hirt fractionation procedure (6) as previously described (3). Unintegrated viral DNAs to be analyzed by gel electrophoresis under denaturing conditions were treated with RNase A in 1 M NaCl to prevent hydrolysis of ribonucleotide linkages in the DNA (3).…”

“…Presoaking and hybridization of DNA immobilized on nitrocellulose filters were performed as previously described (3). Filters were washed and exposed as previously described (3).…”