The Identification and Characterization of Two Promoters and the Complete Genomic Sequence for the Wiskott–Aldrich Syndrome Gene
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文摘
The Wiskott–Aldrich syndrome (WAS) is an X-linked disorder characterized by immunodeficiency, eczema and thrombocytopenia. The gene responsible for WAS was identified through positional cloning, and the function of the encoded protein (WASP) is still the subject of much speculation. WASP is currently thought to be involved in the regulation of actin polymerization in hematopoietic cells. To study the elements that regulate the WASP gene, we have identified the sites for transcription initiation. We found that two promoters were responsible for controlling WASP expression. Multiple transcription initiation sites were found immediately adjacent to the translation start site, however an alternate exon with a second promoter region was identified 6 kb upstream. Examination of the 5′ sequence adjacent to the initiation sites in both promoters failed to reveal a TATA or CCAAT box, but numerous putative transcription factor binding sites including Sp1, Ets, c-Myb and PU.1 were apparent. Reporter constructs generated from each promoter showed functional activity in the Jurkat T-cell and HEL erythro-megakaryocytic cell lines. Although the alternate exon sequence was extremely GC rich and contained several potential binding elements, the primary promoter was stronger than the upstream promoter in the cell lines assayed. The transcription factor binding site profiles within each promoter suggested that they may play different roles in regulating WASP expression depending on the stage of differentiation and development, and the cell lineage. In this study we have also reported the complete nucleotide sequence of the coding and intervening sequences for the WASP gene. A comprehensive knowledge of the genomic structure and the further characterization of WASP gene expression will facilitate the continued investigation of mutations in WAS patients, and the eventual prospect of gene therapy.