Journal:

Article Title: Disruption of Gammaherpesvirus 68 Gene 50 Demonstrates that Rta Is Essential for Virus Replication

doi: 10.1128/JVI.77.10.5731-5739.2003

Figure Lengend Snippet: Genomic structure of gene 50 recombinant viruses. (A) Southern blot analysis. (Left) wt, G50.Stop.[Frt-Kan-Frt], and G50.Stop.Frt BAC viral genomes were purified from E. coli DH10B cells, digested with SpeI, electrophoresed, blotted, and hybridized with a biotin-labeled gene 50 probe (bp 66642 to 69462). The biotin-labeled gene 50 probe was generated by using the Detector random primer DNA biotinylation kit (Kirkegaard & Perry Laboratories), and the Southern blot was developed by using the DNA Detector genomic Southern blotting kit (Kirkegaard & Perry Laboratories) according to the manufacturer's instructions. (Right) wt γHV68 or wt BAC and G50.Stop.Frt BAC (G50 KO in the text) viral genomes were purified from virions isolated from NIH 3T12 and gene 50-expressing stable cell line 14.29, respectively, digested with XbaI, electrophoresed, blotted, and hybridized with a 32P-labeled gene 44 probe (bp 61444 to 62183). On both panels, the fragment sizes of the molecular size markers are shown to the left of each blot (1-kb DNA ladder for the left panel, lambda DNA-BstEII digest for the right panel; New England Biolabs). To the right of each blot are shown the predicted sizes of the viral DNA fragments detected by the respective probes in each blot. (B) Nucleotide sequence of the region containing the mutation in the G50.Stop.Frt BAC. The genome coordinates are to the right of the nucleotide sequence. The nucleotide sequence shown in lowercase letters denotes the gene 50 intron of the spliced gene 50 transcript, and the sequence in uppercase letters denotes the second gene 50 exon of the spliced gene 50 transcript. The splice acceptor site is denoted with an arrowhead. The ORF 50 ATG is boxed. The mutation in G50.Stop.Frt BAC is depicted in boldface, the introduced TAG stop codon is boxed, the FRT site is underlined, and the XbaI site within the FRT site is boxed and italicized.

Article Snippet: The biotin-labeled gene 50 probe was generated by using the Detector random primer DNA biotinylation kit (Kirkegaard & Perry Laboratories), and the Southern blot was developed by using the DNA Detector genomic Southern blotting kit (Kirkegaard & Perry Laboratories) according to the manufacturer's instructions. (Right) wt γHV68 or wt BAC and G50.Stop.Frt BAC (G50 KO in the text) viral genomes were purified from virions isolated from NIH 3T12 and gene 50-expressing stable cell line 14.29, respectively, digested with Xba I, electrophoresed, blotted, and hybridized with a 32 P-labeled gene 44 probe (bp 61444 to 62183).

Techniques: Recombinant, Southern Blot, Purification, Labeling, Generated, Isolation, Expressing, Stable Transfection, Lambda DNA Preparation, Sequencing, Mutagenesis