In a polyomavirus-transformed rat cell line, designated LPT, the polyomavirus DNA is integrated into a single chromosomal site. Treatment of LPT cells with carcinogens induces amplification of the integrated virus DNA and flanking cellular sequences. We show that the amplification is arrested within a specific cell DNA segment that maps 1.3 to 1.85 kilobases beyond one virus-cell DNA junction, defined as-the left junction. We also present the sequence of an 897-base-pair fragment spanning the arrest site. This fragment contains an unusual sequence element, which consists of two contiguous components, a potential cruciform with stems of 6 base pairs and a d(G-A)27 d(T-C)27 tract, and maps 1,497 to 1,564 nucleotides beyond the left junction. The possibility that this unusual sequence plays a role in the arrest of the amplification process is discussed. (6,18,21).We have previously reported experiments in which the extent of the amplified region (amplicon) was determined by a Southern blot analysis of DNAs from uninduced and induced LPT cells (1). The hybridization probe used for the analysis was a cloned 0.95-kilobase (kb) fragment of cell DNA flanking one of the two virus-cell DNA junctions, defined as the left junction. These studies indicated that the amplification is arrested within a specific segment of the cell DNA that maps beyond the 0.95-kb probe (1).Here, we report the results of a Southern blot analysis of LPT DNA, in which two recently cloned hybridization probes derived from more distal regions of the flanking cell DNA were used in addition to the previously available probe. These experiments provided more rigorous proof of the existence of a sharp boundary between the flanking cell DNA, which is strongly amplified, and the more distal cell DNA, which is only slightly amplified. We also report the sequencing of a fragment extending through the boundary. We discovered in this fragment an unusual sequence element consisting of two components, a potential cruciform structure and a d(G-A)27 -d(T-C)27 tract. These components are contiguous, and both map within the boundary region. The possibility that this sequence element plays a role in the arrest of the amplification process is discussed. To induce amplification of the integrated Py DNA and flanking chromosomal sequences in LPT cells, the cells were treated with mitomycin C (MMC), which is the most effective inducing agent in this system (7). To map the amplified flanking sequences, high-molecular-weight chromosomal DNAs were purified from the MMC-treated cells and from LPT cells that were not exposed to MMC. Samples of the two DNAs were digested with various restriction enzymes, and the digests were analyzed by Southern blotting (27). The blots were hybridized with the three probes mentioned above.A physical map of the left portion of the LPT amplicon, the map position of the previously available 0.95-kb probe, * Corresponding author. designated SCL2-1 (1, 22), and the map positions of the two new probes, designated SCL7-2 and SCL7-3, are shown in Fig...