Figure 2

Mapping of K222 proviruses in the human genome. (A) Schematic representation of the primer sets used to isolate K222 by PCR. The genomic structure of a centromeric provirus K111 is shown; the viral genes gag, pro, pol, env, and np9, surrounded by LTRs, integrated into centromeric repeats (CER:D22Z3). The target site duplication of K111 GAATTC is indicated. The primers P1 and P2 bind CER:D22Z3. These primers were used in combination with primers that span the provirus genome. Arrows indicate the position and orientation of the primers; the number above indicates the nucleotide position they bind in reference to K111. Mapping to the 5′ end of the provirus was performed using the primer P1 and a set of HERV-K (HML-2) reverse primers. Mapping to the 3′ end of the provirus was performed with the reverse primer P2 and a set of HERV-K (HML-2) forward primers. (B, C) Isolation of K222 provirus. The sequence of K222 was detected by PCR from DNA of the cell lines H9 and HUT78, which lack K111 5′ end. Normal human DNA, containing K111, was used as a control for the PCR reaction. The number shown for each lane represents the primers. The gels show the amplification products of the 5′ mapping (B) or 3′ mapping (C) of centromeric proviruses in H9, HUT78, and normal human DNA using different combinations of primers. A molecular size ladder is indicated at the left. No amplification products were detected in H9 and HUT78 cell lines, in contrast to normal human DNA, when using the primer sets P1-982R, P1-2499R (B), or primer sets P2-1965F, and P2-2641F (C). An asterisk indicates a band that was shown by sequencing to be the result of non-specific amplification. Sequencing of the mapping products obtained from DNA of H9 and HUT78 cells reveals the sequence of K222.