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A 10.3-kb DNA fragment in the 5'-flanking region of the rat prolactin (rPRL) gene was isolated from F1BGH(1)2C1, a strain of rat pituitary tumor cells (GH cells) that produces prolactin in response to 5-bromodeoxyuridine (BrdU). Following transfection and integration into genomic DNA of recipient mouse L cells, this DNA induced amplification of the adjacent thymidine kinase gene from Herpes simplex virus type 1 (HSV1TK). We confirmed the ability of this "Amplicon" sequence to induce amplification of other linked or unlinked genes in DNA-mediated gene transfer studies. When transferred into the mouse L cells with the 10.3-5'rPRL gene sequence of BrdU-responsive cells, both the human growth hormone and the HSV1TK genes are amplified in response to 5-bromodeoxyuridine. This observation is substantiated by BrdU-induced amplification of the cotransferred bacterial Neo gene. Cotransfection studies reveal that the BrdU-induced amplification capability is associated with a 4-kb DNA sequence in the 5'-flanking region of the rPRL gene of BrdU-responsive cells. These results demonstrate that genes of heterologous origin, linked or unlinked, and selected or unselected, can be coamplified when located within the amplification boundary of the Amplicon sequence.

Bromodeoxyuridine (BrdUrd) treatment of the prolactin nonproducing subclone of GH cells (rat pituitary tumor cells) induces amplification of a 20-kilobase DNA fragment including all of the prolactin gene coding sequences. This amplified DNA segment, which is flanked by two unamplified regions, thus designates a unit of BrdUrd-induced amplified sequence. Cloned DNA segments, 10.3 kilobases long, from the 5' end of the rat prolactin gene of BrdUrd-responsive and -nonresponsive cells, were ligated to the thymidine kinase gene of herpes simplex virus type 1 (HSV1TK), and the hybrid DNA was transferred to thymidine kinase-deficient mouse fibroblast cells by transfection. The HSV1TK gene and the rat prolactin gene were amplified together in drug-treated transfectants carrying the hybrid DNA HSV1TK gene and rat prolactin gene of BrdUrd-responsive GH cells. These results suggest that the 10.3-kilobase DNA segment at the 5' end of the rat prolactin gene of BrdUrd-responsive GH cells carries the information for drug-induced gene amplification (amplicon) and that another gene, such as the HSV1TK gene, is also amplified when the latter is placed adjacent to this segment.