Identification of common virus integrations in the YY1 locus.

(A) Inverse PCR. Genomic DNA from leukemia cells was digested withHhaI. After ligation PCR was performed with primers L1 and L2 followed by a nested PCR with primers L1N and L2N to amplify LTR-flanking fragments from circularized DNA.(B) Nested PCR with LTR and YY1 primers to detect position and orientation of Graffi-1.4 MuLV integration in the YY1 promoter region. The lowercase letters indicate the position at the promoter in base pairs (bp). The first PCR was performed with the primer sets L1, Y1 (a), L1, Y2 (b), L2, Y1 (c), and L2, Y2 (d), followed by a nested PCR with primers L1N, Y1N (a) and L1N, Y2N (b), L2N, Y1N (c), and L2N, Y2N (d). Probes Y1P and Y2P were used to analyze the specificity of the PCR band by Southern blot. (C) Example of Southern blot analysis to determine virus integration and orientation in the 5′ region of the YY1 gene. Results depicted are from DNA samples of leukemias 1, 2, 5, and 6. PCR products from the 4 different primer combinations (a, b, c, and d) were analyzed. In this example, the blot was hybridized with probe Y1P. The presence of the band in lane 1c indicates that tumor 1 has a virus integration in the reverse orientation. Tumor 5 has 2 YY1 virus integrations in the reverse orientation (lane 5c). Tumor 2 has an integration in the forward orientation (lane 2a). Tumor 6 harbors 2 integrations in both orientations (lanes 6a and 6c). All bands were sequenced to determine the exact location of virus integration. (D) Examples of virus integrations and orientations found in the YY1 promoter.