MOUSE 230-KDA BULLOUS PEMPHIGOID ANTIGEN GENE - STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF THE 5'-FLANKING REGION AND INTERSPECIES CONSERVATION OF THE DEDUCED AMINO-TERMINAL PEPTIDE SEQUENCE OF THE PROTEIN

The 230-kDa bullous pemphigoid antigen is a hemidesmosomal protein of the cutaneous basement membrane zone. The primary sequences deduced fr om full-length human cDNAs predict that this molecule consists of a ce ntral rod region and nanking globular domains. To get insight into reg ulation of the 230-kDa bullous pemphigoid antigen gene (BPAG1), and to evaluate evolutionary conservation of the amino-terminus of the prote in, we screened a mouse genomic DNA library with a 0.3-kb cDNA corresp onding to the 5' end of the human 230-kDa bullous pemphigoid antigen c DNA. A positive clone was isolated, and Southern analysis of the clone with the 0.3-kb cDNA allowed isolation of a 3.0-kb Hind III fragment containing the 5' end of the coding sequence. Alignment of the sequenc es of this subclone and human BPAG1 sequences revealed that this fragm ent contained 2466 bp of 5'-flanking DNA, upstream from the ATG transl ation initiation site, and 258 bp of translatable sequences that encod e a putative polypeptide of 86 amino acids at the amino-terminus of th e protein. This deduced polypeptide showed 91% homology with the corre sponding human sequence. The TATAAA and CCAAT consensus sequences, as well as several putative cis-regulatory elements, were identified in t he 5'-flanking region of the mouse DNA. To test the functional promote r activity of the 5'-flanking DNA, three mouse BPAG1 promoter/CAT repo rter gene constructs, with the promoter segments spanning from -1133, -525, and -213 to -1, were developed. Transient transfections of mouse transformed keratinocytes (Pam 212 cells) with these constructs revea led clearly detectable CAT activities, indicating that the 5'-flanking region contains a functional promoter. Furthermore, these experiments suggested that the upstream sequences contain upregulatory elements, as well as elements that confer, at least in part, tissue specificity to the expression of the mouse 230-kDa BPA gene.