cDNA clone for the alpha-chain of human beta-hexosaminidase: deficiency of alpha-chain mRNA in Ashkenazi Tay-Sachs fibroblasts.

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Abstract
A c[complementary]DNA clone was isolated containing sequences complementary to mRNA encoding the .alpha.-chain of the lysosomal enzyme .beta.-hexosaminidase. RNA from a human lung fibroblast strain, IMR90, was enriched for .beta.-hexosaminidase messenger by polysome immunoselection with antiserum against .beta.-hexosaminidase A. This preparation was used to construct cDNA recombinant plasmids by the Okayama-Berg vector primer procedure. After transformation of Escherichia coli, 385 ampicillin-resistant colonies were obtained, 44 of which contained inserts in the plasmid DNA. Differential hybridization, with cDNA probes prepared from polysomal RNA enriched or depleted for .beta.-hexosaminidase messenger, was used to screen the recombinant plasmids for sequences encoding .beta.-hexosaminidase. One clone, p.beta.H.alpha.-1, containing a cDNA insert of .apprxeq. 240 base pairs, was identified in this manner. The plasmid hybrid-selected a messenger from placental RNA that programmed a translation system to synthesize the .alpha.-chain of .beta.-hexosaminidase. p.beta.H.alpha.-1 hybridized to an mRNA of .apprxeq. 1.9 kilobases in preparations enriched separately in messenger for the .alpha.-chain or for both .alpha.- and .beta.-chains (by polysome immunoselection with antiserum against isolated .alpha.-chain or against .beta.-hexosaminidase A, respectively). It did not hybridize to an RNA preparation enriched for messenger of .beta.-chain by immunoselection with antiserum against .beta.-hexosaminidase B. The 1.9-kilobase mRNA was observed in poly(A)+ RNA preparations from control fibroblasts and from fibroblasts of a Tay-Sachs patient that synthesize an altered .alpha.-chain; however, it was not seen in similar preparations from fibroblasts of four Ashkenazi Tay-Sachs patients.