Functional characterization of the 5′ portion of Btk intron 1. (A) The diagram shows the extent of the Btk genomic sequence flanking exon 1 that was subcloned to produce the largest reporter construct pBtkpro+1029. The Sma I site (S) 1.8 kb upstream of exon 1 and the HindIII site (H) 1.0 kb downstream indicate the 5′ and 3′ extents of the subclone, respectively. Also shown are the Sp1 and PU.1 transcription factor binding sites.31,32Restriction sites used to make the deletions included: P,Ppu10I; R, Rsa I; K, Kpn I; and St, StuI. The intron 1 acceptor sequence is shown 5′ to the start of the luciferase gene (LUC). (B) The deletion constructs with the extent of the remaining intron given in base pairs. The final construct represents the fusion between 5′ exon 1 and 3′ exon 15 and the first 119 bp of intron 15. The relative luciferase activity for each construct in the three cell lines tested is given in the table on the right. Reporter construct pBtkpro was assigned the relative activity level of 1 and all other values indicate the fold increase from this basal level. Experiments were performed in duplicate and repeated three times. Values within experiments and between experiments consistently differed by less than 10%.