Selected article for: "cdna amplification and specific amplification"

Title: Isolation, characterization, and expression of cDNAs encoding murine alpha-mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex N-glycans
  • Document date: 1991_12_2
  • ID: qrg1rtzi_4
    Snippet: The most well characterized of the processing a-mannosidases is Man lI. The enzyme has been purified and extensively characterized from rat liver (for review see reference 29) . It is a transmembrane glycoprotein with an apparent molecular mass of 124 kD by SDS-PAGE and a catalytic domain facing the lumen of the Golgi complex. Release of a 110AD catalytically active soluble form of the enzyme can be accomplished by a mild chymotrypsin digestion o.....
    Document: The most well characterized of the processing a-mannosidases is Man lI. The enzyme has been purified and extensively characterized from rat liver (for review see reference 29) . It is a transmembrane glycoprotein with an apparent molecular mass of 124 kD by SDS-PAGE and a catalytic domain facing the lumen of the Golgi complex. Release of a 110AD catalytically active soluble form of the enzyme can be accomplished by a mild chymotrypsin digestion of permeabilized or solubilized Golgi membranes (28) . The chymotrypsin-cleaved form ofthe enzyme has been purified and is catalytically indistinguishable from the intact enzyme (30) , while it differs in NH,-terminal sequence and hydrophobic character. NHz-terminal sequence data from the 110-kD soluble form of the enzyme along with internal peptide sequences have allowed us to generate a Man II-specific cDNA probe by mixed oligonucleotide-primed amplification of cDNA (26) . A partial cDNA clone was isolated from an oligo(dT)-primed rat liver cDNA library which spanned -40% of the Man II open reading frame . We present here the isolation of cDNA clones which span the entire open reading frame and much of the 5' and 3'untranslated regions of Man II from a random-and oligo(dT)-primed murine cDNA library. The open reading frame confirms the map of peptide sequence data from the purified rat protein demonstrating that Man II conforms to the domain structure model common among the Golgi glycosyltransferases . Transient expression of a full length murine Man II cDNA clone in COS cells directs the overexpression of enzyme activity and the synthesis of immunoreactive material in a perinuclear membrane array consistent with the localization of the Man II polypeptide in the Golgi complex of the transfected cells.

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