CLONING AND CHARACTERIZATION OF THE FULL-LENGTH CDNA AND GENOMIC SEQUENCES ENCODING MURINE ACID CERAMIDASE

The full-length cDNA and genomic sequences encoding murine acid cerami dase (AC; E.C. 3.5.1.23) have been isolated and characterized. The 217 6-bp cDNA was similar to 80% identical to the human cDNA (Koch et at, 1996) and predicted a 394-amino-acid polypeptide that was similar to 9 0% identical to the human protein. A fluorescence-based assay system w as developed to determine AC enzymatic activity, and transfection of C OS-1 cells with the full-length mouse cDNA led to increased AC activit y, demonstrating its functionality. The murine AC gene, which spanned similar to 38 kb, consisted of 14 exons separated by 13 introns. The e xons ranged in size from 46 to 1038 bp and were flanked by exon/intron junctions that adhered closely to known donor and acceptor splice sit e consensus sequences. Exon 1 encoded the putative translation start s ite and the signal peptide region, while exon 14 encoded the carboxy e nd of the AC polypeptide and all of the 3' untranslated region. Sequen ce analysis of a 497-bp region upstream from the first in-frame ATG re vealed several features of a housekeeping promoter, as well as several tissue-specific and/or hormone-inducible regulatory sites, Insertion of this sequence into a chloramphenicol acyltransferase (CAT) expressi on vector led an approximately fivefold increase in CAT activity after transfection into NIH3T3 cells. Northern blot analysis and enzymatic assays also were carried out on various murine tissues to examine AC e xpression. Of the tissues studied, the highest AC activity and mRNA le vels were found in the kidney, followed by the brain; almost no AC act ivity or mRNA was found in the testis or skeletal muscle. These latter studies provided clear evidence that despite the housekeeping functio n of AC, its expression was tissue-specific. (C) 1998 Academic Press.