title: Promoters for the human beta-hexosaminidase genes, HEXA and HEXB.
abstract: Human lysosomal beta-hexosaminidases are encoded by two genes, HEXA and HEXB, specifying an alpha- and a beta-subunit, respectively. The subunits dimerize to form beta-hexosaminidase A (alpha beta), beta-hexosaminidase B (beta beta), and beta-hexosaminidase S (alpha alpha). This enzyme system has the capacity to degrade a variety of cellular substrates: oligosaccharides, glycosaminoglycans, and glycolipids containing beta-linked N-acetylglucosaminyl or N-galactosaminyl residues. Mutations in either the HEXA gene or HEXB gene lead to an accumulation of GM2 ganglioside in neurons, resulting in the severe neurodegenerative disorders termed the GM2 gangliosidoses. To identify the DNA elements responsible for hexosaminidase expression, we ligated the 5'-flanking sequences of both the human and mouse hexosaminidase genes to a chloramphenicol acetyltransferase (CAT) gene. The resulting plasmids were transfected into NIH-3T3 cells and CAT activity was determined as a measure of promoter strength. By 5' deletion analysis, it was found that essential sequences for HEXA expression resided within a 40-bp region between 100 bp and 60 bp upstream of the ATG initiation codon. This area contained two potential estrogen response element half-sites as well as potential binding sites for transcription factors NF-E1 and AP-2. Similarly, important HEXB promoter sequences were localized to a 60-bp region between 150 bp and 90 bp upstream of the ATG codon. By performing scanning mutagenesis on a 60-bp region within the 150-bp HEXB construct, we defined an essential promoter element of 12 bp that contained two potential AP-1 sites. The mouse Hexa and Hexb 5'-flanking sequences were found to contain regions similar in sequence, location, and activity to the essential promoter elements defined in the cognate human genes. No sequence similarity was found, however, between 5'-flanking regions of the HEXA and HEXB genes. These essential promoter elements represent potential sites for HEXA and HEXB mutations that could alter enzyme expression in Tay-Sachs and Sandhoff diseases, respectively.
authors.affiliation: Section on Biochemical Genetics, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: F
authors.initials: F
authors.lastname: Norflus
authors.forename: S
authors.initials: S
authors.lastname: Yamanaka
authors.forename: R L
authors.initials: RL
authors.lastname: Proia
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D017930:Genes, Reporter; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D007985:Leydig Cells / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000096:biosynthesis / Q000235:genetics; D018482:Muscle, Skeletal / Q000201:enzymology; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D009928:Organ Specificity; D010920:Placenta / Q000201:enzymology; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012497:Sandhoff Disease / Q000201:enzymology / Q000235:genetics; D012708:Sertoli Cells / Q000201:enzymology; D013045:Species Specificity; D013661:Tay-Sachs Disease / Q000201:enzymology / Q000235:genetics; D014781:Viscera / Q000201:enzymology; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics*
chemical_list: D007527:Isoenzymes; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D001619:beta-N-Acetylhexosaminidases