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a Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Department of Surgery, Harvard Medical School, Boston, Massachusetts 02114
b Division of Immunology and Department of Pathology, Sackler School of Graduate Studies, Tufts University School of Medicine, Boston, Massachusetts 02111
c University of Chicago, Chicago, Illinois 60637
Müllerian-inhibiting substance (MIS) plays an essential role in mammalian male sexual development; thus, it is important to determine how the tightly regulated expression of the MIS gene is transcriptionally controlled. Transcription of eukaryotic genes is dependent on regulatory elements in the enhancer and one or both distinct elements in the core promoter: the TATA box, and the initiator (Inr) element. Because the human MIS gene does not contain a consensus TATA and has not been reported to contain an Inr element, we hypothesized that the initiator region of the core promoter was essential for promoter activity. Transient transfection assays were conducted using an immortalized Embryonic Day 14.5 male rat urogenital ridge cell line (CH34) that expresses low levels of MIS. These studies revealed that promoter activity is dependent on the region around the start site (-6 to +10) but not on the nonconsensus TATA region. Electrophoretic mobility shift assays demonstrated that the human MIS initiator sequence forms a specific DNA-protein complex with CH34 cell nuclear extract, HeLa cell nuclear extract, and purified TFII-I. This complex could be blocked or supershifted by the addition of antibodies directed against TFII-I. These data suggest that the human MIS gene contains a functional initiator that is specifically recognized by TFII-I.
1 Supported by NIH grant HD30812 to P.K.D. and in part by ACS grant RPG-98-104-01-TBE and NIH grant AI41147 to A.L.R., NIH grant HD08177 and a fellowship from ACS to T.R.C., NIH grant HD33462 to M.A.W., the Gerald Austen Surgical Research and Surdna GAR funds to N.M., and the M.G.H. Medical Discovery Award to C.M.H.
2 Correspondence: Patricia K. Donahoe, Pediatric Surgical Research Laboratories, Warren 10, Massachusetts General Hospital, 32 Fruit St., Boston, MA 02114. FAX: 617 726 5057;donahoe.patricia{at}mgh.harvard.edu
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