Isolation of cDNA clones coding for the beta subunit of human beta-hexosaminidase.

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Abstract
The major forms of .beta.-hexosaminidase (2-acetamido-2-deoxy-.beta.-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) occur as multimers of .alpha. and .beta. chains-hexosaminidase A (.alpha..beta.a.beta.b) and hexosaminidase B 2(.beta.a.beta.b). To facilitate the investigation of .beta.-chain biosynthesis and the nature of mutation in Sandhoff disease, a human hexosaminidase .beta.-chain c[complementary]DNA clone was isolated. Hexosaminidase B (10 mg) was treated with CNBr, 5 peptide fragments were isolated by reverse-phase HPLC, and their amino acid sequences were determined. One of these contained a string of 6 amino acids from which an oligonucleotide probe was defined. The SV40-transformed human fibroblast cDNA library of Okayama and Berg was screened by colony hybridization with the radiolabeled probe. Probe-binding clones (13) were selected out of 50,000 clones screened. Four of these designated pHex were shown to be identical at their 3'' ends by restriction enzyme mapping, differing only in their 5'' extensions (1.4-1.7 kilobases). The nucleotide sequence of a 174-base-pair segment contained the deduced amino acid sequence of 2 of the 5 CNBr peptides, indicating that the pHex clones encode the .beta. subunit of hexosaminidase. pHex cDNA was found homologous to multiple bands in digests of genomic human DNA totaling 43 kilobases (kb), all of which were mapped to chromosome 5 in somatic cell hybrids, as expected of the HEXB gene. The pHex cDNA also hybridized to a 2.2-kilobase RNA that apparently codes for the pre-.beta.-polypeptide of hexosaminidase. This RNA species was absent in the fibroblasts of 1 of 3 patients with Sandhoff disease examined. These clones will be of value to diagnosis and carrier detection of Sandhoff disease in affected families.