Genomic analysis of TβR-I in JK cells. (A) Schematic depicting the PCR primers and Southern blotting probe used to analyze the intron-exon structure surrounding exons 1 and 2 of the TβR-I gene. Shown is exons 1 and 2 of TβR-I (stippled boxes) and their intervening intron. The hatched box of exon 1 represents the 178-bp deletion identified by RT-PCR analysis of JK cell mRNA, and the thick bar underlying this region depicts the ³²P-radiolabeled PCR fragment used in Southern blotting experiments. Amplification of exon 1 from genomic DNA was accomplished using primer pair 1 and 2 to amplify the normal 255-bp product. Exon 1 and 2 initiated amplification across intron 1 was accomplished using primer pair 3 and 4 or primer pair 5 and 6 on genomic DNA obtained from cultured cells or patient biopsies, respectively. Primer sequences and PCR reaction conditions are described in Materials and Methods. (B) PCR of genomic DNA identifies an ∼5 kb deletion in JK cell TβR-I gene. (Upper panel) A total of 100 ng of genomic DNA from JK, HepG2 hepatoma (Hep), HT1080 fibrosarcoma (HT), or MDA-MB-231 breast cancer (231) cells was subjected to PCR amplification using primer pair 1 and 2 that flanked exon 1 of the human TβR-I. The resulting PCR products were fractionated through a 2.5% agarose/TAE gel as described in Materials and Methods. Data shown is a representative picture of an ethidium bromide-stained gel demonstrating the presence of the 255-bp TβR-I product only in TGF-β–responsive cells. (Lower panel) One microgram of genomic DNA from JK, HepG2 (Hep), HT1080 (HT), or MDA-MB-231 (231) cells was subjected to PCR amplification using primer pair 3 and 4, which flanked the 178-bp deletion in the JK cell TβR-I cDNA. Shown is a representative picture of an ethidium bromide-stained gel demonstrating the presence only in JK cells of a single amplified PCR product, whose size (260 bp) and sequence was identical to that of the JK cell TβR-I cDNA. (C) Radiolabeled deletion fragment fails to hybridize with JK cell genomic DNA. Ten micrograms of genomic DNA from JK, HepG2 (Hep), HT1080 (HT), or MDA-MB-231 (231) cells was digested overnight at 37°C with Hind III, and after transfer to nylon membrane, hybridized with a ³²P-radiolabeled probe corresponding to the 178-bp sequence absent in JK cell TβR-I cDNA as described in Materials and Methods. Data shown is a representative autoradiograph demonstrating the presence of a hybridizing signal in the genomes of TGF-β–responsive cells, but not in JK cells. (D) Detection of mutated TβR-I in tumor biopsies obtained before establishment of the JK cell line. JK cell genomic DNA (Gen), cDNA, or genomic DNA obtained from patient tumor biopsies performed in 1989 and 1996 were subjected to PCR amplification using primer pair 5 and 6, which flanked the 178-bp deletion in the JK cell TβR-I cDNA. Shown is a representative picture of an ethidium bromide-stained gel demonstrating the presence of mutated TβR-I gene in the patient’s primary tumor samples before the establishment of the JK cell line.