Integration of mini-retroviral DNA: a cell-free reaction for biochemical analysis of retroviral integration.
After retroviral infection of a permissive cell, the viral RNA is reverse-transcribed to make a DNA copy of the viral genome. Integration of this DNA copy into the host genome is a necessary step for efficient viral replication. We have developed a cell-free system for integration of exogenous mini-retroviral DNA. The termini of this linear mini-Moloney murine leukemia virus (MoMLV) DNA are designed to mimic the ends of authentic unintegrated MoMLV DNA. The viral proteins required for integration can be provided either as a cytoplasmic extract of MoMLV-infected NIH 3T3 cells or as disrupted MoMLV particles. Phage lambda DNA serves as the target for integration. Genetic markers present on the mini-MoMLV DNA enable integration events to be detected, and the recombinants recovered, by selection in Escherichia coli. Integration, which occurs at heterogeneous locations in the target DNA, is absolutely dependent on the presence of a source of viral proteins and a divalent cation in the reaction mixture. The fidelity of the integration reaction was confirmed by sequencing the junctions between the integrated MoMLV DNA and adjacent lambda DNA sequence. In each case, as expected for authentic MoMLV DNA integration, a 4-base-pair duplication of target DNA sequence flanked the integrated MoMLV DNA.
Fujiwara
T
T
Laboratory of Molecular Biology, National Institute of Diabetes, and Digestive and Kidney Diseases, Bethesda, MD 20892.
Craigie
R
R
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002474:Cell-Free System; D002850:Chromatography, Gel; D003593:Cytoplasm / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics*; D019426:Integrases; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics*; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
D004279:DNA, Viral; D014764:Viral Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases