cDNA clone for the alpha-chain of human beta-hexosaminidase: deficiency of alpha-chain mRNA in Ashkenazi Tay-Sachs fibroblasts.
We have isolated a cDNA clone 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 approximately equal to 240 base pairs, was identified in this manner. The plasmid hybrid-selected a messenger from placental RNA that programed a translation system to synthesize the alpha-chain of beta-hexosaminidase. p beta H alpha-1 hybridized to an mRNA of approximately equal to 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.
D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology; D006596:Hexosaminidases / Q000172:deficiency / Q000235:genetics*; D006801:Humans; D008168:Lung / Q000196:embryology; D046911:Macromolecular Substances; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D013661:Tay-Sachs Disease / Q000201:enzymology*; D001619:beta-N-Acetylhexosaminidases
D046911:Macromolecular Substances; D012333:RNA, Messenger; D004247:DNA; D006596:Hexosaminidases; D001619:beta-N-Acetylhexosaminidases