Sequence analysis and DNA-protein interactions within the 5 flanking region of the Ca2+/calmodulin-dependent protein kinase II alpha-subunit gene.

摘要

The 5' flanking region of the brain Ca2+/calmodulin-dependent protein kinase II alpha-subunit gene was identified and characterized. A total of 430 bases was sequenced upstream from the translation initiation codon, and the site of transcription initiation was located at -149 or -147 bases as determined by primer extension and S1 nuclease protection analysis, respectively. TATA and CAAT boxes were absent from their standard positions; however, the 5' flanking region was rich in G + C and contained a GGGCG and a TATATAA sequence 76 and 160 bases upstream from the transcription initiation site, respectively. Moreover, the sequence CAACGG was found 85 and 146 bases upstream from this site, indicating presumptive binding sites for the Myb protein. Gel-mobility shift assays revealed that a 120-base-pair fragment, which included the G + C-rich, TATA, and CAACGG sequences bound nuclear proteins specifically. DNA-protein complexes with similar gel mobilities were obtained with nuclear extracts from rat forebrain or cerebellum and from neonatal or adult brains. Extracts from rat liver, kidney, and spleen generated specific DNA-protein complexes with different electrophoretic mobilities, suggesting the occurrence of different nuclear proteins that bind to 5' regulatory elements of the Ca2+/calmodulin-dependent kinase II alpha-subunit gene.