Journal: Journal of Virology
Article Title: Murine Gammaherpesvirus 68 Lacking Thymidine Kinase Shows Severe Attenuation of Lytic Cycle Replication In Vivo but Still Establishes Latency
Figure Lengend Snippet: Genomic structure of TK + and TK − viruses. (A) The TK − Kan + virus was generated by inserting a FRT-flanked kanamycin resistance gene at genomic coordinates 33301 to 33450. The kanamycin resistance coding sequence was removed by Flp recombination to leave an inset of 167 bp (TK − Kan − ). This virus was reverted by homologous recombination with an unmutated genomic segment (TK + R). An independent mutant (TK − Del) was generated by RecA-mediated recombination of BAC DNA with a genomic plasmid clone in which genomic coordinates 33293 to 34300 are deleted by digestion with Avr II and Pme I. (B) Viral DNA was digested with Xba I, Eco RI, or Sac I, electrophoresed, blotted, and probed with a 32 P-labeled genomic Eco RI fragment spanning the TK locus.
Article Snippet: The central portion (33293 to 34300) of the TK open reading frame (ORF) (32879 to 34813) was excised by digestion with Avr II and Pme I, blunting with T4 DNA polymerase (New England Biolabs, Hitchin, United Kingdom), and religation with T4 DNA ligase (New England Biolabs).
Techniques: Generated, Sequencing, Homologous Recombination, Mutagenesis, BAC Assay, Plasmid Preparation, Labeling