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Characterization of the Murine Pituitary Tumor Transforming Gene (PTTG) and Its Promoter¹

Cedars-Sinai Research Institute, University of California School of Medicine, Los Angeles, California 90048

Address all correspondence and requests for reprints to: Shlomo Melmed, M.D., Academic Affairs 2015, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California 90048. E-mail: melmed{at}csmc.edu

We recently isolated rat pituitary tumor transforming gene (PTTG) complementary DNA and showed its potent in vitro and in vivo transforming activity. We now characterize the mouse PTTG gene and its promoter. The entire gene is composed of five exons and four introns and spans about 7 kb. Northern analysis showed that PTTG was expressed in several tumor cell lines examined, but not in all normal tissues, implying a correlation between PTTG and tumorigenesis. Using rapid amplification of 5'-cDNA ends, the transcription start site was localized at -303 nucleotides upstream to the ATG codon in both F9 and AtT20 cells. An approximately 4.3-kb upstream region demonstrated promoter activity in AtT20 cells as well as other cell lines tested, and in vivo, the cloned promoter driving an enhanced green fluorescent protein transgene exhibited transcriptional activation in testis and embryo. Serial deletions showed that -313 bp of the 5'-flanking region was critical for promoter activity. Three elements contribute to promoter activity. Both element A (-313/-293) and element C (-180/-160), in an electrophoretic mobility shift assay using NIH-3T3 nuclear extract, formed three specific complexes, which were competed by a known Sp1 oligo; one complex was supershifted by Sp1 antibody, and the other two complexes were both supershifted by an Sp3 antibody. Two mutants disrupting element A resulted in up to 70% loss of promoter activity and abrogated formation of specific DNA-protein binding complexes, implying a more important role for element A. Element B (-249/-229) shows more than 80% homology to a consensus c-myb element, but formed two specific complexes that differed from that of c-myb in the electrophoretic mobility shift assay. Thus, the integrity and possible cooperation among these elements contribute to the basal promoter activity of the mouse PTTG oncogene homolog.

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